Fgf21 compositions for treatment or prevention of neovascularization of the eye and methods therefor

ABSTRACT

The instant disclosure provides methods and compositions related to discovery of a long-acting FGF21 as a therapeutic target for treatment or prevention of neovascular eye diseases or disorders that are characterized by angiogenesis, or of vascular diseases of the eye. Therapeutic and/or prophylactic uses and compositions of long-acting FGF21 are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of U.S. Provisional Application62/453,352 filed on Feb. 1, 2017 and U.S. Provisional Application62/595,917 filed on Dec. 7, 2017. The entire contents of theseapplications are incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under NIH EY024864,EY017017, EY022275, and HD18655. The government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

Pathological neovascularization, a leading cause of blindness, is seenin retinopathy of prematurity, diabetic retinopathy and age relatedmacular degeneration. There is currently no cure for retinopathy ofprematurity, diabetic retinopathy, and age related macular degeneration.Therefore, there is an unmet need for treatments of retinopathy ofprematurity, diabetic retinopathy, and age related macular degenerationthat ameliorate and/or prevent pathological neovascularization.

SUMMARY OF THE INVENTION

The instant disclosure relates, at least in part, to the discovery ofFGF21 as a therapeutic target for pathologic vessel growth in patientswith neovascular eye diseases including retinopathy of prematurity,diabetic retinopathy and age-related macular degeneration. In certainaspects, stable and/or stabilized forms of FGF21 (i.e., long-actingFGF21) are used to treat or prevent neovascular eye diseases. In certainaspects of the disclosure, it is also identified that targeting of theFGF21 pathway as described herein can exert a therapeutic effect forneurovascular diseases of the eye such as diabetic retinopathy,retinopathy of prematurity (ROP), retinitis pigmentosa (RP), age-relatedmacular degeneration (AMD) and macular telangiectasia (MacTel). Thedisclosure provides a method for treating or preventing neovascular eyediseases in a subject, the method involving (a) identifying a subjecthaving or at risk of neovascular eye disease; and (b) administering along-acting FGF21 composition to the subject, thereby treating orpreventing neovascular eye disease in the subject.

In one aspect, the disclosure provides a method for treating orpreventing neovascularization and/or angiogenesis in the eye of asubject, the method involving identifying a subject having or at risk ofneovascularization and/or angiogenesis in the eye; and administering apharmaceutical composition including a stabilized FGF21 agent to thesubject, thereby treating or preventing neovascularization and/orangiogenesis in the eye of the subject.

In one embodiment, the subject has or is at risk of developingneovascular retinopathy. In another embodiment, the subject has or is atrisk of developing diabetic retinopathy in Type I or Type II diabetes,retinopathy of prematurity (ROP), retinitis pigmentosa (RP) and/ormacular telangiectasia (MacTel).

In another embodiment, choroidal neovascularization and/or angiogenesisis treated or prevented.

Optionally, retinal neovascularization and/or angiogenesis in theretinal cells of the eye is treated or prevented.

In one embodiment, the stabilized FGF21 agent comprises an FGF21polypeptide conjugated to an antibody scaffold. Optionally, the FGF21polypeptide is a modified FGF21. In certain embodiments, the modifiedFGF21 is dHis/Ala129Cys, optionally the modified FGF21 is conjugated atCys 129 to the antibody scaffold. Optionally, two or more FGF21polypeptide molecules are conjugated to one antibody scaffold the two ormore FGF21 polypeptide molecules can be the same or different from eachother. In some embodiments, the antibody scaffold is a CovX-2000scaffold.

In certain embodiments, the stabilized FGF21 agent is a long actingFGF21 analog, optionally PF-05231023.

In one embodiment, the stabilized FGF21 agent possesses a half-life ofat least 1.5× the half-life of a native FGF21 peptide when assayed forstability under identical conditions, optionally at least 2× thehalf-life of a native FGF21 peptide, optionally at least 3× thehalf-life of a native FGF21 peptide, optionally at least 4× thehalf-life of a native FGF21 peptide, optionally at least 5× thehalf-life of a native FGF21 peptide, optionally at least 8× thehalf-life of a native FGF21 peptide, optionally at least 10× thehalf-life of a native FGF21 peptide, optionally at least 12× thehalf-life of a native FGF21 peptide, optionally at least 15× thehalf-life of a native FGF21 peptide, optionally at least 20× thehalf-life of a native FGF21 peptide, optionally at least 30× thehalf-life of a native FGF21 peptide, optionally at least 40× thehalf-life of a native FGF21 peptide, optionally at least 50× thehalf-life of a native FGF21 peptide, optionally at least 60× thehalf-life of a native FGF21 peptide or optionally at least 70× thehalf-life of a native FGF21 peptide.

In certain embodiments, the stabilized FGF21 agent possesses a half-lifeof at least 0.8 h in the circulation of a mammal, optionally at least 1h, at least 2 h, at least 3 h, at least 4 h, at least 5 h, at least 7 h,at least 10 h, at least 15 h, at least 20 h, at least 25 h, at least 28h or at least 30 h in the circulation of a mammal, optionally whereinthe mammal is human.

In one embodiment, the pharmaceutical composition is administered to theeye of the subject, optionally by intravitreal injection.

In some embodiments, the pharmaceutical composition is administered inan amount sufficient to increase retinal levels of APN in the subject.

Another aspect of the disclosure provides a pharmaceutical compositionfor use in treating or preventing neovascularization and/or angiogenesisin the eye of a subject that includes a stabilized FGF21 agent and apharmaceutically acceptable carrier.

An additional aspect of the disclosure provides for use of a stabilizedFGF21 agent in the preparation of a medicament for treatment orprevention of neovascular retinopathy, diabetic retinopathy in type Idiabetes, retinopathy of prematurity (ROP), retinitis pigmentosa (RP) ormacular telangiectasia (MacTel) in a subject.

A further aspect of the disclosure provides a method of treating asubject suffering from diabetic retinopathy, retinitis pigmentosa orage-related macular degeneration involving administering an effectiveamount of a long acting FGF-21 agent to the subject.

In one embodiment, the age-related macular degeneration is wet maculardegeneration or dry macular degeneration.

In another embodiment, the diabetic retinopathy is proliferativediabetic retinopathy or non-proliferative diabetic retinopathy.

In an additional embodiment, the retinitis pigmentosa is autosomalrecessive, autosomal dominant or X-linked.

In certain embodiments, the long acting FGF-21 agent is two ΔHis1/A129Cmodified FGF-21 peptides each conjugated to a Fab region of a humanizedIgG1k mAb.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this disclosure belongs. The following references provide one ofskill with a general definition of many of the terms used in thisdisclosure: The Cambridge Dictionary of Science and Technology (Walkered., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.),Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionaryof Biology (1991). As used herein, the following terms have the meaningsascribed to them below, unless specified otherwise.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

An “agent” is meant any small compound, antibody, nucleic acid molecule,or peptide or fragment thereof.

As used herein, “Age-related macular degeneration,” or “AMD” refers toan eye condition which causes a deterioration or breakdown of themacula, a small spot near the center of the retina and the part of theeye needed for sharp central vision. More specifically, thephotoreceptor cells within the macula die off slowly, thus accountingfor the progressive loss of vision.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

An “agonist” as used herein is a molecule which enhances the biologicalfunction of a protein. The agonist may thereby bind to the targetprotein to elicit its functions. However, agonists which do not bind theprotein are also envisioned. The agonist may enhance the biologicalfunction of the protein directly or indirectly. Agonists which increaseexpression of certain genes are envisioned within the scope ofparticular embodiments of the disclosure. Suitable agonists will beevident to those of skill in the art. For the present disclosure it isnot necessary that the agonist enhances the function of the targetprotein directly. Rather, agonists are also envisioned which stabilizeor enhance the function of one or more proteins upstream in a pathwaythat eventually leads to activation of targeted protein. Alternatively,the agonist may inhibit the function of a negative transcriptionalregulator of the target protein, wherein the transcriptional regulatoracts upstream in a pathway that eventually represses transcription ofthe target protein.

An “antagonist” may refer to a molecule that interferes with theactivity or binding of another molecule, for example, by competing forthe one or more binding sites of an agonist, but does not induce anactive response.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

By “effective amount” is meant the amount of an agent required toameliorate the symptoms of a disease relative to an untreated patient.The effective amount of active agent(s) used to practice the presentdisclosure for therapeutic treatment of a disease varies depending uponthe manner of administration, the age, body weight, and general healthof the subject. Ultimately, the attending physician or veterinarian willdecide the appropriate amount and dosage regimen. Such amount isreferred to as an “effective” amount.

The phrase “longer half-life” has its ordinary meaning as understood bypersons of skill in the art. Merely by way of example, and by no meansas a limitation on the meaning of the term, the following description ofthe term is informative: any appreciable increase in the length of timein which FGF21 may be circulating and able to bind beta klotho and FGF21receptors either in vivo or in vitro as compared to the half-life of thenative FGF21 alone either in vivo or in vitro.

The term “macula” refers to a small area within the retina. The maculais the part of the retina that is responsible for central vision,allowing things to be seen clearly. Although only a small part of theretina, the macula is more sensitive to detail than the rest of theretina. Many older people develop macular degeneration as part of thebody's natural aging process. Symptoms of macular degeneration includeblurriness, dark areas or distortion in central vision, or evenpermanent loss in central vision. It usually does not affect side orperipheral vision.

The term “neovascularization” refers to the formation of functionalmicrovascular networks with red blood cell perfusion. Neovascularizationdiffers from angiogenesis in that angiogenesis is mainly characterizedby the protrusion and outgrowth of capillary buds and sprouts frompre-existing blood vessels. Choroidal neovascularization is theformation of new microvasculature within the innermost layer of thechoroid of the eye.

As used herein, “obtaining” as in “obtaining an agent” includessynthesizing, purchasing, or otherwise acquiring the agent.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

By “photoreceptor cells” refers to the bulk of neurons in the retina.The photoreceptor cells capture light energy units (photons) andregister the events as electrical signals of the central nervous system.The signals are then relayed to intermediary layers of neurons in theretina that process and organize the information before it istransmitted along the optic nerve fibers to the brain.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

By “reference” is meant a standard or control, e.g., a standard orcontrol condition.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%,75%, or 100%.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

As used herein, a “stable FGF21” or “stabilized FGF21” refers to acomposition that includes an active FGF21 polypeptide and a modification(as compared to a WT, unmodified form of FGF21) that has the effect ofproducing a form of FGF21 agent that is more protease stable than acorresponding WT, unmodified form of FGF21 (i.e., than native FGF21). Astabilized FGF21 composition of the instant disclosure therefore possessa greater half-life than native FGF21, when assessed for stability in anart-recognized stability assay (e.g., an in vitro stability assay, e.g.,in the presence of one or more proteases and/or and in vivo stabilityassay, e.g., when administered to the plasma, blood, saliva, etc. of asubject). Samples are analyzed for the levels of FGF21 using an ELISA in96-well microtiter plate coated with an anti-FGF21 mAb.

In certain embodiments, a “stable FGF21” or “stabilized FGF21” of theinstant disclosure can exhibit biological activity over an extendedperiod of time, as compared to a WT, unmodified form of FGF21. Forexample, a “stable FGF21” or “stabilized FGF21” of the instantdisclosure can exhibit biological activity (e.g., a therapeutic and/orprophylactic effect) in a subject to which such stable FGF21 isadministered over a period of an hour or more, two hours or more, threehours or more, four hours or more, five hours or more, ten hours ormore, twenty hours or more, a day or more, two days or more, three daysor more, four days or more, five days or more, a week or more, two weeksor more, or even a month or more after a single administration of the“stable FGF21” or “stabilized FGF21”. Optionally, a single dose ormultiple doses of the “stable FGF21” or “stabilized FGF21” can beadministered to a subject, optionally to achieve dosage to a subjectwithin a desired (e.g., therapeutically effective) range over anextended duration of time.

A “therapeutically effective amount” is an amount sufficient to effectbeneficial or desired results, including clinical results. An effectiveamount can be administered in one or more administrations.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms (e.g., AMD,MacTel or other angiogenesis-associated disease or disorder of the eye,or of tumors in general) associated therewith. It will be appreciatedthat, although not precluded, treating a disorder or condition does notrequire that the disorder, condition or symptoms associated therewith becompletely eliminated.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

Other features and advantages of the disclosure will be apparent tothose skilled in the art from the following detailed description andclaims

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict micrographs and charts that demonstrate FGF21treatment (particularly stabilized/long-acting FGF21 treatment)decreased hypoxia-induced retinal neovascularization, whereas FGF21deficiency increased hypoxia-induced retinal neovascularization.Quantification of neovascularization in P17 retinal whole mounts wasperformed in mouse oxygen-induced retinopathy (OIR). FIG. 1A showsmicrographs and an accompanying chart demonstrating the extent ofneovascularization in retinas treated with native FGF21 (nFGF21), or along-acting FGF21 analog (PF-05231023), or with vehicle control. n=14-19retinas per group. One-way ANOVA was performed upon raw results,followed by Bonferroni's multiple comparisons post test. n.s.=nosignificance. FIG. 1B shows micrographs demonstrating observedneovascularization and quantifying fold changes observed for suchneovascularization in retinas of Fgf21^(+/+) and Fgf21^(−/−) mice.n=12-13 retinas per group. Unpaired t test. Whole mount vessels werestained with isolectin (red), and neovascularization was pseudo-coloredwhite. Representative images are shown. Scale bar, 1 mm Data werepresented as mean±SEM. Fold-changes were calculated in comparison to thecontrol group.

FIGS. 2A-2F depict micrographs, charts, and schematics demonstratingthat FGF21 suppressed retinal neovascularization via adiponectin (APN)to reduce TNFα. Quantification of neovascularization in P17 retinalwhole mounts was performed after oxygen-induced retinopathy. In wholemounts, vessels were stained with isolectin (red) and neovascularizationwas pseudo-colored white. Representative images are shown. Scale bar, 1mm Data was presented as mean±SEM. Unpaired t test. n.s., nosignificance. Fold of change was calculated. FIG. 2A depicts a graphshowing relative mRNA levels in WT P17 normal retinas for FGF21receptors Fgfr1, Fgfr2, Fgfr3, Fgfr4 and co-receptor β-klotho (Klb)(n=6-8 pooled retinas per group). FIG. 2B depicts a graph demonstratingthat in WT P17 hypoxic retinas, long-acting FGF21 agent PF-05231023exerted an inducing effect on retinal APN (n=6 pooled retinas pergroup). PF-05231023 was administered from P12-16. FIG. 2C depictsmicrographs and charts demonstrating that Apn^(−/−) mice exhibitedincreased levels of neovascularization, as compared to WT mice (n=17-20retinas per group). FIG. 2D depicts micrographs and charts demonstratingthat Apn^(−/−) mice treated with PF-05231023 did not show a significantresponse to PF-05231023 treatment (n=11-14 retinas per group). FIG. 2Edepicts graphs demonstrating the effect of PF-05231023 treatment on Tnfαlevels in WT or Apn^(−/−) retinas (n=6-8 pooled retinas per group),where WT mice were clearly responsive to PF-05231023 treatment andApn^(−/−) retinas exhibited an ablated response. FIG. 2F depicts aschematic of the perceived role of FGF21 modulation in neovascularretinas.

FIGS. 3A-3D depict micrographs and charts demonstrating that exogenousFGF21 decreased retinal neovascularization in Vldlr^(−/−) mice. FIG. 3Adepicts 3 dimensional (3D) reconstruction of retinal neovesselsextending from the outer plexiform layer (OPL) towards the retinalpigment epithelium (RPE). PF-05231023 (i.p. injected daily from P8-15)decreased retinal neovessel extension towards RPE seen with isolectin(red) stained vessels. FIG. 3B depicts representative images ofisolectin-stained vessels in retinal whole mounts, and neovessels(lesions) are highlighted in white (bottom) (with photoreceptor layerfacing up). Scale bar, 1 mm FIG. 3C shows the result of quantifyingnumber and area (size) of vascular lesions (n=11-18 retinas per group).FIG. 3D demonstrates that in Vldlr^(−/−) retinas, PF-0523102 exertedsignificant effects on both Apn and Tnfα levels (n=4-6 pooled retinasper group). Data was presented as mean±SEM. Unpaired t test.Fold-changes were calculated.

FIGS. 4A-4D depicts micrographs, charts, and schematics demonstratingthat FGF21 administration decreased choroidal neovascularization inmice. FIG. 4A depicts a schematic showing an approach for laser-inducedchoroidal neovascularization in mice.

FIG. 4B depicts representative images of isolectin-stained choroidalneovessels, in both vehicle- and PF-05231023-treated mice. The area ofinduced lesions was quantified (n=6-8 mice per group). Scale bar: 200 μm(top); 50 μm (bottom). FIG. 4C depicts charts that demonstrate theeffect of PF-05231023 on Apn and Tnfα levels in neovascularchoroid-retina complexes (n=4-6 pooled retinas per group). FIG. 4Ddepicts charts that demonstrate the effect of PF-05231023 on Vegfa inthe three mouse models of neovascular eye diseases examined (n=6-8pooled retinas per group). Data were presented as mean±SEM. Unpaired ttest. n.s., no significance. Fold-changes were calculated.

FIG. 5 depicts micrographs and charts demonstrating that intra-vitrealPF-05231023 administration induced a trend of reduction inneovascularization in OIR. In WT mice after P12 intra-vitrealPF-05231023, a trend of reduction of neovascularization was observed.The contralateral eye was injected with vehicle. (n=7 retinas pergroup). Unpaired t test. See also FIGS. 1A-1B.

FIGS. 6A-6D depict graphs demonstrating that FGF21 promoted normalretinal revascularization. Quantification of revascularization in P17retinal whole mounts after oxygen-induced retinopathy was performed.FIG. 6A depicts WT retinas treated with PF-05231023 or vehicle controlfrom P12-P16 (n=14-19 retinas per group). Unpaired t test. FIG. 6Bdepicts Fgf21^(+/+) and Fgf21^(−/−) retinas (n=12-13 retinas per group).Unpaired t test. FIG. 6C depicts WT and Apn^(−/−) mice (n=17-20 retinasper group). Unpaired t test. FIG. 6D depicts Apn^(−/−) mice treated withPF-05231023 (n=11-14 retinas per group). Unpaired t test.

FIGS. 7A-7D depict micrographs and charts demonstrating that FGF21receptors and adiponectin colocalized in retinal neovessels. FIG. 7Adepicts laser-capture microdissected retinal layers and vessels fromnormal (N) and oxygen-induced retinopathy (OIR) P17 retinas;localization and quantitation of Fgfr1 and Fgfr3 was performed usingqPCR (n=6-8 pooled retinas). Unpaired t test. Scale bar: 20 μm. FIG. 7Bdepicts immunohistochemistry for adiponectin (green) and lectin (red) inP17 OIR retinal whole mounts. Scale bar: 40 μm. FIG. 7C depicts theeffect of PF-05231023 (left) and adipoRon (right) on migrated area inHRMEC would healing assays. One-way ANOVA. FIG. 7D depicts effect ofPF-05231023 (left) and adipoRon (right) on HRMEC cell viablity. One-wayANOVA.

FIGS. 8A-8B depict micrographs and graphs demonstrating the observedimpacts of FGF21 deficiency. FIG. 8A depicts that FGF21 deficiencydelayed normal retinal vascular development in neonatal mice. FIG. 8Bdepicts that FGF21 deficiency worsened hyperglycemic retinopathy inneonatal mice.

FIGS. 9A-9B show micrographs and graphs demonstrating that FGF21promoted retinal vascular development in hyperglycemic mouse neonates.FIG. 9A shows the effects observed following administration of nativeFGF21 (nFGF21). FIG. 9B depicts the effects observed afteradministration of long-acting FGF21 (PF-05231023). As shown,administration of long-acting FGF21 improved retinal vascular growth.Unpaired t test.

FIGS. 10A-10B depict micrographs and graphs demonstrating thatlong-acting FGF21 protection against hyperglycemic retinopathy wasdependent on adiponectin (APN). FIG. 10A demonstrates that APNdeficiency completely abolished long-acting FGF21 effects that wouldotherwise have been observed upon treatment of hyperglycemicretinopathy. FIG. 10B shows that FGF21 administration increasedhigh-molecular-weight (HMW) and hexamer APN levels in serum. Unpaired ttest.

FIG. 11 demonstrates that long-acting FGF21 protection against thedecrease in cone function in retinal degenerating mice (Rd10) modelingretinitis pigmentosa.

FIG. 12 depicts a polypeptide sequence and structure of an exemplarylong-acting FGF21 polypeptide, as depicted in Huang et al., 2013 anddescribed elsewhere herein (SED ID NO.: 1). The sequence of recombinanthuman ΔHis FGF21 (A129C) and a schematic of the bivalent FGF21CovX-Body, CVX-343, is shown. The underlined C residue corresponds toposition 129, where the FGF21 protein is conjugated to the antibodyscaffold.

FIGS. 13A-13D depict graphs showing that serum FGF21 levels weredecreased in Akita mice. FIG. 13A shows serum FGF21 levels measured byELISA. n=6-8 mice per group. FIGS. 13B-13D shows results from qPCR forFgf21, Fgfr1, β-klotho expression in diabetic WT and Akita retinas.n=3-4 mice per group. Unpaired t test.

FIGS. 14A-14E depict a series of schematic illustrations and graphsshowing that PF-05231023 administration improved retinal function indiabetic Akita mice. FIG. 14A is a schematic illustration of PF-05231023treatment in 7-to-8-month-old Akita mice. 10 mg/kg PF-05231023 was i.p.injected twice a week for a month. ERG was measured before and aftertreatment. ERG plots with ‘white’ (for maximal a-wave) and “green” (formaximal b-wave) light stimulation are shown to demonstrate theparameters: a-wave (photoreceptors), b-wave (bipolar cells),oscillitatory potentials (OPs, inner retinal neurons). Photoreceptoramplitude (Rm_(P3)) and sensitivity (S), bipolar cell response amplitude(Rm_(P2)) and sensitivity (1/K_(P2)), inner retinal neuronal saturatingenergy (Em) and sensitivity (1/i_(1/2Em)), as well as total retinalsensitivity (Sm) were measured and calculated. FIG. 14B showsrepresentative ERG plots in 7-to-8-month-old WT mice (black),age-matched Akita mice before (blue) and after (orange) PF-05231023administration. FIG. 14C shows overall changes in different ERGparameters in WT mice, Akita mice before and after PF-05231023administration. FIG. 14D shows a comparison of retinal sensitivity (Sm)in WT mice, Akita mice before and after PF-05231023 administration. n=5to 10 mice per group. ANOVA followed by Tukey″s test. FIG. 18E showsplots of ERG parameters in 7-to-8-month-old Akita mice before and afterPF-05231023 administration. n=5 mice per group. Paired t test. Data waspresented as Mean±SEM.

FIGS. 15A-15E depict a series of plots photographs and graphs showingthat PF-05231023 administration restored the retinal morphology in Akitamice. FIG. 15A shows the correlation of post-receptor cell sensitivity(1/K_(P2)) with the sum of changes in photoreceptor sensitivity(S_(Rod)) and saturated amplitude (R_(Rod)) in Akita mice. n=10 eyes pergroup. Pearson r test. FIG. 15B shows results from qPCR of Arrestin4 andRhodopsin in age-matched WT mice and Akita mice treated with eithervehicle or PF-05231023. n=3-4 mice per group. ANOVA. FIGS. 15C-15D arephotographs showing the immunohistochemistry of cones (cone arrestin,red), rods (rhodopsin, green) and nuclei (DAPI, blue) in age-matched WTmice and Akita mice treated with either vehicle or PF-05231023. GCL,ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.Scale bar: 50 μm. FIG. 19E shows an optical coherence tomography (OCT)for photoreceptor inner and outer segment in age-matched wild-type (WT)mice (black line) and Akita mice treated with either vehicle (blue line)or PF-05231023 (orange line). n=4-10 mice per group. *P<0.05, **P<0.01.ANOVA.

FIGS. 16A-16B depict graphs showing that PF-05231023 administrationdecreased IL-1β expression in diabetic retinas. FIG. 16A shows resultsfrom qPCR for pro-inflammatory markers (IL-1β, Vegfa, Tnfα, IL-6) andanti-inflammatory markers (IL-10, Apn) in WT and Akita mouse retinas.n=3-4 mice per group. FIG. 16B shows results from Akita mouse retinastreated with vehicle (PBS) or PF-05231023. n=3-4 mice per group.Unpaired t test. Data is presented as Mean±SEM.

FIGS. 17A-17E depict a series of graphs and photographs of Western blotsshowing that PF-05231023 administration induced NRF2 levels byactivating the AKT pathway and decreased photoreceptor-derived IL-1β.FIG. 17A is a Western blot of p-AKT, AKT, NRF2 in non-diabetic WT,diabetic Akita with vehicle or PF-05231023 administration for one month.β-ACTIN was used as internal control. n=3 mice per group. ANOVA. FIG.17B show results from qPCR of IL-1β in cone photoreceptors in vitro(661W). Oxidative stress was induced with 0.5 mM paraquat (PQ) for 1hour. The culture medium was changed and cells were treated with 500ng/ml PF-05231023 or vehicle for 24 hours. n=4 independent replicates.ANOVA followed by Bonferroni's multiple comparisons test. FIG. 17C showsresults from qPCR of Nrf2 and Nfκb in 661W with PQ-induced oxidativestress followed by treatment with 500 ng/ml PF-05231023 or vehicle for24 hours. n=4 independent replicates. ANOVA followed by Bonferroni'smultiple comparisons test. FIG. 17D shows results from qPCR of Nrf2 in661W with PQ-induced oxidative stress followed by co-treatment with 500ng/ml PF-05231023 and perifosine at 5 or 10 μM. ANOVA. FIG. 17E is aWestern blot of NRF2, p-NFκB and NFκB in 661W with PQ-induced oxidativestress followed by treatment with 500 ng/ml PF-05231023 or vehicle for24 hours. Protein lysate was isolated from 661W. β-ACTIN was used asinternal control. Lane only with loading dye was negative control. n=4independent replicates. ANOVA followed by Bonferroni's multiplecomparisons test.

FIGS. 18A-18I depict a series of plots and graphs showing thatPF-05231023 administration protected the retinal function in STZ-induceddiabetic mice, independent of APN. FIG. 18A is a schematic ofSTZ-induced type 1 diabetes in C57BL/6J (WT) mice. STZ was i.p. injectedin 6-to-8-week-old WT mice (Day1-2: 60 mg/kg; Day3-5: 55 mg/kg). ERG wascompared in diabetic mice before and after PF-05231023 treatment (10mg/kg i.p., twice a week). FIG. 18B depicts representative ERG plots of7-to-8-month-old WT control mice, and WT diabetic mice before and afterPF-05231023 administration. FIG. 18C depicts the overall changes indifferent ERG parameters in WT normal mice, WT diabetic mice before andafter PF-05231023 administration. FIG. 18D depicts a comparison ofretinal sensitivity (Sm) in WT normal mice, WT diabetic mice before andafter PF-05231023 administration. n=3 to 5 mice per group. ANOVAfollowed by Tukey's test. FIG. 18E are plots of retinal sensitivity (Sm)in 7-to-8-month-old WT diabetic mice before and after PF-05231023administration. n=3 mice per group. Paired t test. FIG. 18F showrepresentative ERG plots of 7-to-8-month-old Apn diabetic mice beforeand after PF-05231023 administration. FIG. 18G shows the overall changesin different ERG parameters in Apn diabetic mice before and afterPF-05231023 administration. FIG. 18H show plots of retinal sensitivity(Sm) in Apn^(−/−) diabetic mice before and after PF-05231023administration. n=3 mice per group. Paired t test. FIG. 18I are graphsshowing results from qPCR for IL-1β in diabetic WT and Apn^(−/−) mouseretinas treated with vehicle (PBS) or PF-05231023. n=3 mice per group.Unpaired t test. ERG plots with ‘white” and “green” light stimulationare shown. Data was presented as Mean±SEM.

FIG. 19 is a schematic of a flow chart for PF-05231023 protectionagainst DR in type 1 diabetic mice. Schematic of signaling pathway thatPF-05231023 may improve retinal neurovascular activity in diabetic miceby activating the AKT pathway and inducing anti-oxidative NRF2, which inturn decreases photoreceptor-derived pro-inflammatory marker IL-1β.

FIG. 20 depicts a graph showing FGF21 receptor Fgfr1 expression inretinal neuronal layers. RGC (retinal ganglion cells), INL (innernuclear layer), and ONL (outer nuclear layer) were isolated usinglaser-captured microdissection and RNA was extracted. qPCR of Fgfr1 wasconducted.

FIGS. 21A-21C depict graphs showing that PF-05231023 administration didnot change body weight (FIG. 21A), blood glucose levels (FIG. 21B) orserum triglycerides (FIG. 21C) in Akita mice. Body weight was decreased,blood glucose levels and serum triglyceride (TG) levels were induced indiabetic Akita mice versus normal WT mice; PF-05231023 administrationlowered blood glucose levels, but did not change the body weight andserum TG levels in Akita mice. Data is presented as Mean±SEM. n=5-12mice per group. ANOVA followed by Bonferroni's multiple comparisonstest. n.s., no significance.

FIG. 22 is a series of plots and Comparison of ERG parameters in Akitamice measured at 7-month and 8-months of age. n=3 mice per group. ANOVAfollowed by Tukey's test. Representative ERG plot is shown. Data waspresented as Mean±SEM.

FIGS. 23A-23C are graphs showing that blood glucose levels (FIG. 23B)were increased, body weight (FIG. 23A) and serum TG levels (FIG. 23C)were not changed in STZ-induced diabetic WT mice versus normal WT mice;PF-05231023 administration did not change the body weight, blood glucoselevels or serum TG levels in STZ-induced diabetic WT mice. Data ispresented as Mean±SEM. n=3-5 mice per group. ANOVA followed byBonferroni's multiple comparisons test. n.s., no significance.

FIGS. 24A-24C are graphs showing results from a qPCR of retinal Fgf21(FIG. 24A), Fgfr1 (FIG. 24B) and Klb (FIG. 24C) in Akita mice withPF-05231023 or vehicle treatment. n=3-4 mice per group. Unpaired t test.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure is based, at least in part, upon the discovery of FGF21as a therapeutic target for pathologic vessel growth in patients withneovascular eye diseases including retinopathy of prematurity, diabeticretinopathy and age-related macular degeneration. In certain aspects ofthe disclosure, stable forms of FGF21 (i.e., long-acting FGF21) are usedto treat neovascular eye diseases. In certain aspects of the disclosure,it is also identified that targeting of the FGF21 pathway as describedherein can exert a therapeutic effect for neurovascular diseases of theeye such as diabetic retinopathy, retinopathy of prematurity, retinitispigmentosa, age-related macular degeneration (AMD) and maculartelangiectasia (MacTel). The disclosure provides a method for treatingor preventing neovascular eye diseases in a subject, the methodinvolving (a) identifying a subject having or at risk of neovascular eyedisease; and (b) administering a long-acting FGF21 composition to thesubject, thereby treating or preventing neovascular eye disease in thesubject.

Pathological neovascularization, a leading cause of blindness, is seenin retinopathy of prematurity, diabetic retinopathy and age relatedmacular degeneration. Using a mouse model of hypoxia-driven retinalneovascularization, oxygen-induced retinopathy, it was discovered thatfibroblast growth factor 21 (FGF21) administration suppressed, and FGF21deficiency worsened, retinal neovessel growth. The protective effect oflong-acting FGF21 against neovessel growth was abolished in adiponectin(APN)-deficient mice, and long-acting FGF21 administration alsodecreased neovascular lesions in two models of neovascular age-relatedmacular degeneration, very-low-density-lipoprotein-receptor-deficientmice having retinal angiomatous proliferation and laser-inducedchoroidal neovascularization. Long-acting FGF21 inhibited Tnfαexpression but did not alter Vegfa expression in neovascular eyes. Asdescribed herein, FGF21 (particularly long-acting FGF21) is therebydescribed as a therapeutic modality for pathologic vessel growth inpatients having neovascular eye diseases including retinopathy ofprematurity, diabetic retinopathy and age-related macular degeneration.

Additional aspects and embodiments of the invention are described below.

Mechanism of Action

Without wishing to be bound by theory, the instant disclosure isbelieved to function in the following manner Pathological retinalneovessel growth is a major cause of vision loss in retinopathy ofprematurity in premature infants, in macular telangiectasia, diabeticretinopathy and age-related macular degeneration in adults. Uncontrolledneovessel growth is driven by the need for oxygen and for energysubstrates (Hellstrom et al., 2013; Joyal et al., 2016). Currenttreatments for retinal neovascularization have limitations. Althoughlaser photocoagulation helps preserve central vision, it causesperipheral visual loss (Ciulla et al., 2003). In addition, anti-vascularendothelial growth factor (VEGF) drugs effectively treatneovascularization in some, but not all patients and there are safetyconcerns about the long-term effects including degeneration of normalblood vessels, neural retina and choroid (Arevalo, 2013; Cheung et al.,2012; Fernando Arevalo, 2013; Osaadon et al., 2014; Sato et al., 2012).Therefore, better therapeutic agents are needed for the effectivetreatment of vision-threatening neovascularization.

As described herein, FGF21 suppresses retinal and choroidal ocularpathologic angiogenesis in three different mouse models of disease. Inhumans, long-acting FGF21 analog administration increases circulatingadiponectin (APN) in a dose-dependent manner (Gaich et al., 2013;Talukdar et al., 2016). In mice, FGF21 administration increases APNproduction to modulate glucose and lipid metabolism (Holland et al.,2013; Lin et al., 2013). Low circulating APN levels may contribute tothe development of neovascular eye diseases in humans (Fu et al., 2016;Fu et al., 2015; Kaarniranta et al., 2012; Mao et al., 2012; Omae etal., 2015). APN administration inhibits retinal and choroidalneovascularization in rodents (Higuchi et al., 2009; Lyzogubov et al.,2012). As described herein, FGF21 inhibits pathological retinal andchoroidal angiogenesis in the eye and FGF21 administration improvesneovascular eye diseases. As described herein, FGF21 has roles in 1)hypoxia-induced neovascular retinopathy (Smith et al., 1994); 2) retinalneovascularization driven by energy deficiency (Joyal et al., 2016); and3) laser-induced choroidal neovascularization (Gong et al., 2015).

The compositions and methods of the instant disclosure are expresslycontemplated for treatment and/or prevention of at least the followingdisorders and/or diseases of the eye, either alone or in combination.

Choroidal Neovascularization (CNV)

Choroidal neovascularization (CNV) is the creation of new blood vesselsin the choroid layer of the eye. CNV can occur rapidly in individualswith defects in Bruch's membrane, the innermost layer of the choroid. Itis also associated with excessive amounts of vascular endothelial growthfactor (VEGF). In addition to wet macular degeneration, CNV can alsooccur frequently with the rare genetic disease pseudoxanthoma elasticumand rarely with the more common optic disc drusen. CNV has also beenassociated with extreme myopia or malignant myopic degeneration, wherein choroidal neovascularization occurs primarily in the presence ofcracks within the retinal (specifically) macular tissue known as lacquercracks. CNV can create a sudden deterioration of central vision,noticeable within a few weeks. Other symptoms which can occur includecolour disturbances, and metamorphopsia (distortions in which straightlines appears wavy). Hemorrhaging of the new blood vessels canaccelerate the onset of symptoms of CNV. CNV may also include thefeeling of pressure behind a subject's eye.

Age-Related Macular Degeneration (AMD)

AMD is a common eye condition and a leading cause of vision loss amongpeople age 50 and older. It causes damage to the macula, a small spotnear the center of the retina and the part of the eye needed for sharp,central vision, which lets us see objects that are straight ahead. Insome people, AMD advances so slowly that vision loss does not occur fora long time. In others, the disease progresses faster and may lead to aloss of vision in one or both eyes. As AMD progresses, a blurred areanear the center of vision is a common symptom. Over time, the blurredarea may grow larger or you may develop blank spots in your centralvision. Objects also may not appear to be as bright as they used to be.

AMD by itself does not lead to complete blindness, with no ability tosee. However, the loss of central vision in AMD can interfere withsimple everyday activities, such as the ability to see faces, drive,read, write, or do close work, such as cooking or fixing things aroundthe house.

Macula

The macula is made up of millions of light-sensing cells that providesharp, central vision. It is the most sensitive part of the retina,which is located at the back of the eye. The retina turns light intoelectrical signals and then sends these electrical signals through theoptic nerve to the brain, where they are translated into the images wesee. When the macula is damaged, the center of your field of view mayappear blurry, distorted, or dark.

MacTel (macular telangiectasia)

Macular telangiectasia is a disease in which the macula is affected,causing a loss of central vision. The macula is a small area in theretina (the light-sensitive tissue lining the back of the eye) that isresponsible for central vision, allowing fine details to be seenclearly. Macular telangiectasia develops when there are problems withthe tiny blood vessels around the fovea, the center of the macula. Thereare two types of macular telangiectasia (Type 1 and Type 2), and eachaffects the blood vessels differently. Macular telangiectasia may occuras a result of a retinal vascular disease or a systemic disease such asdiabetes or hypertension, but in many cases, clinical findings reveal noknown cause.

One serious complication of macular telangiectasia is the development ofabnormal blood vessels under the retina. This is called choroidalneovascularization, and may call for injections of a drug calledvascular endothelial growth factor inhibitors (anti-VEGF). Anti-VEGFmedication targets a specific chemical in the eye that causes abnormalblood vessels to grow under the retina. That chemical is called vascularendothelial growth factor, or VEGF. Blocking VEGF with medicationinjections reduces the growth of abnormal blood vessels, slows theirleakage, helps to reduce swelling of the retina, and in some cases mayimprove vision.

Type 1 Macular Telangiectasia

In Type 1 macular telangiectasia, the blood vessels become dilatedforming tiny aneurysms, causing swelling and damaging macular cells. Thedisease almost always occurs in one eye, which differentiates it fromType 2.

Type 2 Macular Telangiectasia

The most common form of macular telangiectasia is Type 2 maculartelangiectasia, in which the tiny blood vessels around the fovea leak,become dilated (widen), or both. It is a bilateral disease of unknowncause, which characteristic alterations of macular capillary network andneurosensory atrophy. In some cases, new blood vessels form under theretina and they can also break or leak. Fluid from leaking blood vesselscauses the macula to swell or thicken, a condition called macular edema,which affects central vision. Also, scar tissue can sometimes form overthe macula and the fovea, causing loss of detail vision. Type 2 affectsboth eyes but not necessarily with the same severity.

Retinopathy of Prematurity (ROP)

Retinopathy of prematurity (ROP) is a potentially blinding eye disorderthat primarily affects premature infants weighing about 2¾ pounds (1250grams) or less that are born before 31 weeks of gestation (A full-termpregnancy has a gestation of 38-42 weeks). The smaller a baby is atbirth, the more likely that baby is to develop ROP. This disorder—whichusually develops in both eyes—is one of the most common causes of visualloss in childhood and can lead to lifelong vision impairment andblindness. ROP was first diagnosed in 1942.

With advances in neonatal care, smaller and more premature infants arebeing saved. These infants are at a much higher risk for ROP. Not allbabies who are premature develop ROP. There are approximately 3.9million infants born in the U.S. each year; of those, about 28,000 weigh2¾ pounds or less. About 14,000-16,000 of these infants are affected bysome degree of ROP. The disease improves and leaves no permanent damagein milder cases of ROP. About 90 percent of all infants with ROP are inthe milder category and do not need treatment. However, infants withmore severe disease can develop impaired vision or even blindness. About1,100-1,500 infants annually develop ROP that is severe enough torequire medical treatment. About 400-600 infants each year in the USbecome legally blind from ROP.

ROP is classified in five stages, ranging from mild (stage I) to severe(stage V):

Stage I—Mildly abnormal blood vessel growth. Many children who developstage I improve with no treatment and eventually develop normal vision.The disease resolves on its own without further progression.Stage II—Moderately abnormal blood vessel growth. Many children whodevelop stage II improve with no treatment and eventually develop normalvision. The disease resolves on its own without further progression.Stage III—Severely abnormal blood vessel growth. The abnormal bloodvessels grow toward the center of the eye instead of following theirnormal growth pattern along the surface of the retina. Some infants whodevelop stage III improve with no treatment and eventually developnormal vision. However, when infants have a certain degree of Stage IIIand “plus disease” develops, treatment is considered. “Plus disease”means that the blood vessels of the retina have become enlarged andtwisted, indicating a worsening of the disease. Treatment at this pointhas a good chance of preventing retinal detachment.Stage IV—Partially detached retina. Traction from the scar produced bybleeding, abnormal vessels pulls the retina away from the wall of theeye.Stage V—Completely detached retina and the end stage of the disease. Ifthe eye is left alone at this stage, the baby can have severe visualimpairment and even blindness.Most babies who develop ROP have stages I or II. However, in a smallnumber of babies, ROP worsens, sometimes very rapidly. Untreated ROPthreatens to destroy vision.

Infants with ROP are considered to be at higher risk for developingcertain eye problems later in life, such as retinal detachment, myopia(nearsightedness), strabismus (crossed eyes), amblyopia (lazy eye), andglaucoma. In many cases, these eye problems can be treated orcontrolled.

ROP occurs when abnormal blood vessels grow and spread throughout theretina, the tissue that lines the back of the eye. These abnormal bloodvessels are fragile and can leak, scarring the retina and pulling it outof position. This causes a retinal detachment. Retinal detachment is themain cause of visual impairment and blindness in ROP.

Several complex factors may be responsible for the development of ROP.The eye starts to develop at about 16 weeks of pregnancy, when the bloodvessels of the retina begin to form at the optic nerve in the back ofthe eye. The blood vessels grow gradually toward the edges of thedeveloping retina, supplying oxygen and nutrients. During the last 12weeks of a pregnancy, the eye develops rapidly. When a baby is bornfull-term, the retinal blood vessel growth is mostly complete (Theretina usually finishes growing a few weeks to a month after birth). Butif a baby is born prematurely, before these blood vessels have reachedthe edges of the retina, normal vessel growth may stop. The edges of theretina—the periphery—may not get enough oxygen and nutrients.

It is believed that the periphery of the retina then sends out signalsto other areas of the retina for nourishment. As a result, new abnormalvessels begin to grow. These new blood vessels are fragile and weak andcan bleed, leading to retinal scarring. When these scars shrink, theypull on the retina, causing it to detach from the back of the eye.

To date, the most effective proven treatments for ROP are laser therapyor cryotherapy. Laser therapy “burns away” the periphery of the retina,which has no normal blood vessels. With cryotherapy, physicians use aninstrument that generates freezing temperatures to briefly touch spotson the surface of the eye that overlie the periphery of the retina. Bothlaser treatment and cryotherapy destroy the peripheral areas of theretina, slowing or reversing the abnormal growth of blood vessels.Unfortunately, the treatments also destroy some side vision. This isdone to save the most important part of our sight—the sharp, centralvision we need for “straight ahead” activities such as reading, sewing,and driving.

Both laser treatments and cryotherapy are performed only on infants withadvanced ROP, particularly stage III with “plus disease.” Bothtreatments are considered invasive surgeries on the eye, and doctorsdon't know the long-term side effects of each.

In the later stages of ROP, other treatment options include:

-   -   Scleral buckle. This involves placing a silicone band around the        eye and tightening it. This keeps the vitreous gel from pulling        on the scar tissue and allows the retina to flatten back down        onto the wall of the eye. Infants who have had a sclera buckle        need to have the band removed months or years later, since the        eye continues to grow; otherwise they will become nearsighted.        Sclera buckles are usually performed on infants with stage IV or        V.    -   Vitrectomy. Vitrectomy involves removing the vitreous and        replacing it with a saline solution. After the vitreous has been        removed, the scar tissue on the retina can be peeled back or cut        away, allowing the retina to relax and lay back down against the        eye wall. Vitrectomy is performed only at stage V.

While ROP treatment decreases the chances for vision loss, it does notalways prevent it. Not all babies respond to ROP treatment, and thedisease may get worse. If treatment for ROP does not work, a retinaldetachment may develop. Often, only part of the retina detaches (stageIV). When this happens, no further treatments may be needed, since apartial detachment may remain the same or go away without treatment.However, in some instances, physicians may recommend treatment to try toprevent further advancement of the retinal detachment (stage V). If thecenter of the retina or the entire retina detaches, central vision isthreatened, and surgery may be recommended to reattach the retina.

Diabetic Retinopathy

Diabetic retinopathy describes a diabetic eye disease that affects bloodvessels in the retina and is both the most common cause of vision lossamong people with diabetes and the leading cause of vision impairmentand blindness among working-age adults. Chronically high blood sugarfrom diabetes is associated with damage to the blood vessels in theretina, thereby leading to diabetic retinopathy. This changes thecurvature of the lens and results in the development of symptoms ofblurred vision. The blurring of distance vision as a result of lensswelling will subside once the blood sugar levels are brought undercontrol. Better control of blood sugar levels in patients with diabetesalso slows the onset and progression of diabetic retinopathy. Symptomsof diabetic retinopathy may include seeing spots or floaters in asubject's field of vision, blurred vision, having a dark or empty spotin the center of a subject's vision, and difficulty seeing well atnight. Diabetic retinopathy may progress through four stages:

-   -   I. Mild nonproliferative retinopathy: small areas of swelling in        the retinal blood vessels causing tiny bulges, called        microaneurysms to protrude from their walls may occur.    -   II. Moderate nonproliferative retinopathy: progression of the        disease may lead to blood vessels swelling and distorting,        therefore affecting their ability to transport blood.    -   III. Severe nonproliferative retinopathy: more blood vessels        become blocked, depriving the blood supply to areas of the        retina.    -   IV. Proliferative diabetic retinopathy: advanced stage of the        disease where growth factors secreted by the retina trigger the        proliferation of new blood vessels, which grow along the inside        surface of the retina and into the fluid that fills the eye. The        fragility of the new blood vessels makes them more likely to        leak and bleed. Scar tissue can cause retinal detachment        (pulling away of the retina from underlying tissue). Retinal        detachment can lead to permanent vision loss.

Retinitis Pigmentosa (RP)

Retinitis pigmentosa (RP) is an inherited, degenerative eye disease thatcauses severe vision impairment due to the progressive degeneration ofthe rod photoreceptor cells in the retina. This form of retinaldystrophy manifests initial symptoms independent of age—diagnosis occursfrom early infancy to late adulthood. Patients in the early stages of RPfirst notice compromised peripheral and dim light vision due to thedecline of the rod photoreceptors. The progressive rod degeneration islater followed by abnormalities in the adjacent retinal pigmentepithelium (RPE) and the deterioration of cone photoreceptor cells. Asperipheral vision becomes increasingly compromised, patients experienceprogressive “tunnel vision” and eventual blindness. Afflictedindividuals may additionally experience the accumulation of bonespicules in the fundus, defective light-dark adaptations, and nyctalopia(night blindness).

The initial retinal degenerative symptoms of Retinitis Pigmentosa arecharacterized by decreased night vision (nyctalopia) and the loss of themid-peripheral visual field. The rod photoreceptor cells, which areresponsible for low-light vision and are orientated in the retinalperiphery, are the retinal processes affected first during non-syndromicforms of this disease. Visual decline progresses relatively quickly tothe far peripheral field, eventually extending into the central visualfield as tunnel vision increases. Visual acuity and color vision canbecome compromised due to accompanying abnormalities in the conephotoreceptor cells, which are responsible for color vision, visualacuity, and sight in the central visual field. The progression ofdisease symptoms occurs in a symmetrical manner, with both the left andright eyes experiencing symptoms at a similar rate.

A variety of indirect symptoms characterize Retinitis Pigmentosa alongwith the direct effects of the initial rod photoreceptor degenerationand later cone photoreceptor decline. Phenomena such as photophobia,which describes the event in which light is perceived as an intenseglare, and photopsia, the presence of blinking or shimmering lightswithin the visual field, often manifest during the later stages of RP.Findings related to RP have often been characterized in the fundus ofthe eye as the Ophthalamic triad. This includes the development of amottled appearance of the retinal pigment epithelium (RPE) caused bybone spicule formation, a waxy appearance of the optic nerve, and theattentuation of blood vessels in the retina.

A variety of retinal molecular pathway defects have been matched tomultiple known RP gene mutations. Mutations in the rhodopsin gene, whichis responsible for the majority of autosomal-dominantly inherited RPcases, disrupts the rod-opsin protein essential for translating lightinto decipherable electrical signals within the phototransductioncascade of the central nervous system. Defects in the activity of thisG-protein-coupled receptor are classified into distinct classes thatdepend on the specific folding abnormality and the resulting molecularpathway defects. The Class I mutant protein's activity is compromised asspecific point mutations in the protein-coding amino acid sequenceaffect the pigment protein's transportation into the outer segment ofthe eye, where the phototransduction cascade is localized. Additionally,the misfolding of Class II rhodopsin gene mutations disrupts theprotein's conjunction with 11-cis-retinal to induce proper chromophoreformation. Additional mutants in this pigment-encoding gene affectprotein stability, disrupt mRNA integrity post-translationally, andaffect the activation rates of transducin and opsin optical proteins.Additionally, animal models suggest that the retinal pigment epitheliumfails to phagocytose the outer rod segment discs that have been shed,leading to an accumulation of outer rod segment debris. In mice that arehomozygous recessive for retinal degeneration mutation, rodphotoreceptors stop developing and undergo degeneration before cellularmaturation completes. A defect in cGMP-phosphodiesterase has also beendocumented; this leads to toxic levels of cGMP.

Fibroblast Growth Factor 21 (FGF21)

Fibroblast growth factor 21 is a protein that in mammals is encoded bythe FGF21 gene. The protein encoded by this gene is a member of thefibroblast growth factor (FGF) family and specifically a member of the“endocrine” subfamily which includes FGF23 and FGF15/19. FGF familymembers possess broad mitogenic and cell survival activities and areinvolved in a variety of biological processes including embryonicdevelopment, cell growth, morphogenesis, tissue repair, tumor growth andinvasion. FGFs act through a family of four FGF receptors. Binding iscomplicated and requires both interaction of the FGF molecule with anFGF receptor and binding to heparin through an heparin binding domain.Endocrine FGFs lack a heparin binding domain and thus can be releasedinto the circulation. FGF21 action through one of the FGF21 receptorsthus requires interaction with a co-receptor, designated β-klotho.

FGF21 is specifically induced by HMGCS2 activity. The oxidized form ofketone bodies (acetoacetate) in a cultured medium also induced FGF21,possibly via a SIRT1-dependent mechanism. HMGCS2 activity has also beenshown to be increased by deacetylation of lysines 310, 447, and 473 viaSIRT3 in the mitochondria. While FGF21 is expressed in numerous tissues,including liver, brown adipose tissue, white adipose tissue andpancreas, circulating levels of FGF21 are derived specifically from theliver in mice. In liver FGF21 expression is regulated by PPARα andlevels rise substantially with both fasting and consumption of ketogenicdiets. LXR represses FGF21 in humans via an LXR response element locatedfrom −37 to −22 bp on the human FGF21 promoter.

FGF21 stimulates glucose uptake in adipocytes. This effect is additiveto the activity of insulin. FGF21 treatment of adipocytes is associatedwith phosphorylation of FRS2, a protein linking FGF receptors to theRas/MAP kinase pathway. FGF21 injection in ob/ob mice results in anincrease in Glut1 in adipose tissue. FGF21 also protects animals fromdiet-induced obesity when overexpressed in transgenic mice and lowersblood glucose and triglyceride levels when administered to diabeticrodents. Treatment of animals with FGF21 results in increased energyexpenditure, fat utilization and lipid excretion.

Serum FGF-21 levels were significantly increased in patients with type 2diabetes mellitus (T2DM) which may indicate a role in the pathogenesisof T2DM. Elevated levels also correlate with liver fat content innon-alcoholic fatty liver disease and positively correlate with BMI inhumans suggesting obesity as a FGF21-resistant state.

FGF21 stimulates phosphorylation of fibroblast growth factor receptorsubstrate 2 and ERK1/2 in the liver. Acute FGF21 treatment inducedhepatic expression of key regulators of gluconeogenesis, lipidmetabolism, and ketogenesis including glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, 3-hydroxybutyrate dehydrogenase type 1, andcarnitine palmitoyltransferase 1α. In addition, injection of FGF21 wasassociated with decreased circulating insulin and free fatty acidlevels. FGF21 treatment induced mRNA and protein expression of PGC-1α,but in mice PGC-1α expression was not necessary for the effect of FGF21on glucose metabolism.

In mice FGF21 is strongly induced in liver by prolonged fasting viaPPAR-alpha and in turn induces the transcriptional coactivator PGC-1αand stimulates hepatic gluconeogenesis, fatty acid oxidation, andketogenesis. FGF21 also blocks somatic growth and sensitizes mice to ahibernation-like state of torpor, playing a key role in eliciting andcoordinating the adaptive starvation response. FGF21 expression is alsoinduced in white adipose tissue by PPAR-gamma, which may indicate italso regulates metabolism in the fed state.

Activation of AMPK and SIRT1 by FGF21 in adipocytes enhancedmitochondrial oxidative capacity as demonstrated by increases in oxygenconsumption, citrate synthase activity, and induction of key metabolicgenes. The effects of FGF21 on mitochondrial function requireserine/threonine kinase 11 (STK11/LKB1), which activates AMPK.Inhibition of AMPK, SIRT1, and PGC-1α activities attenuated the effectsof FGF21 on oxygen consumption and gene expression, indicating thatFGF21 regulates mitochondrial activity and enhances oxidative capacitythrough an LKB1-AMPK-SIRT1-PGC-1α-dependent mechanism in adipocytes,resulting in increased phosphorylation of AMPK, increased cellular NAD+levels and activation of SIRT1 and deacetylation of SIRT1 targets PGC-1αand histone 3.

Stabilized FGF21

Modifications to FGF21 stabilize the protein and increase the half-lifecompared to native FGF21 without any loss in efficacy or potency. Astabilized or long-acting FGF21, as described herein, comprises an FGF21polypeptide conjugated to an antibody scaffold. In certain embodiments,the modified FGF21 is dHis/Ala129Cys, optionally the modified FGF21 isconjugated at Cys 129 to the antibody scaffold. Optionally, two or moreFGF21 polypeptide molecules are conjugated to one antibody scaffold—thetwo or more FGF21 polypeptide molecules can be the same or differentfrom each other. In some embodiments, the antibody scaffold is ahumanized IgG1K monoclonal antibody. In other embodiments, the antibodyscaffold is the Fab region of a humanized IgG1K monoclonal antibody. Insome embodiments, the antibody scaffold is a CovX-2000 scaffold. Incertain embodiments, the stabilized FGF21 agent is a long acting FGF21analog, optionally PF-05231023.

In some embodiments, a stabilized FGF21 agent (i.e., long-acting FGF21)possesses a half-life of at least 1.5×, at least 2×, at least 3×, atleast 4×, at least 5×, at least 8×, at least 10×, at least 12×, at least15×, at least 20×, at least 30×, at least 40×, at least 50×, at least60×, or at least 70× the half-life of a native FGF21 peptide, whenassayed for stability under identical conditions. In other embodiments,a stabilized FGF21 agent (i.e., long-acting FGF21) possesses a half-lifeof at least 0.8 h, at least 1 h, at least 2 h, at least 3 h, at least 4h, at least 5 h, at least 6 h, at least 7 h, at least 10 h, at least 15h, at least 20 h, at least 25 h, at least 28 h or at least 30 h in thecirculation of a mammal, optionally wherein the mammal is human.

As described herein, the stability or half-life of a modified FGF21(e.g., long-acting FGF21) composition may be determined by assessing thedegree of resistance of such a composition to protease digestion, ascompared to native FGF21 compositions, in vitro, in vivo, or both.Optionally, stability may be assessed by collecting blood and/or serumfrom subjects previously administered a candidate long-acting FGF21agent and detecting the presence of the long-acting FGF21 agent withanti-FGF21 antibodies in such samples at different time points. Serumsamples may be prepared and analyzed using an ELISA-based assay in whichlong-acting FGF21 compounds are captured in 96 well microtiter platescoated with anti-FGF21 mAb followed by detection with ananti-hIgG-horseradish peroxidase (HRP). Alternatively, the activity of acandidate long-acting FGF21 agent can also be assessed as a proxy forthe continued presence of the long-acting FGF21 agent in the sample, atone or more time points post-administration.

Exemplary forms of long-acting FGF21 can be found in the art, forexample as described in U.S. Pat. No. 9,163,277, issued Oct. 20, 2015;U.S. Pat. No. 8,722,622, issued May 13, 2014; U.S. Provisional PatentApplication 61/644,831, filed May 9, 2012; Huang et al., (2013) Journalof Pharmacology and Experimental Therapeutics, 346:270-280; Weng et al.,(2015) PLOS One 10(3) e0119104:1-18; Thompson et al., (2016) J.Pharmacokinet Pharmacodyn 43:411-425; and Talukdar et al., (2016) CellMetabolism 23: 427-440. The disclosure of each of these publications isincorporated herein by reference in its entirety.

In certain embodiments, as depicted in FIG. 12, the long-acting FGF21comprises the following polypeptide sequence (Huang et al., 2013):

(SEQ ID NO: 1) PIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPCPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS..

For example, a provided long acting FGF21 may be a disclosed FGF21peptide sequence such as the above, covalently attached to a combiningsite of an antibody or an antigen binding portion of a scaffold antibody(such as a humanized IgG1k mAb, or e.g., an aldolase catalytic antibody)via a chemical linker (e.g.,3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-[2-(2-(3-oxo-3-((4-(3-oxo-3-(2-oxoazetidin-1-yl)propyl)phenyl)amino)propoxy)ethoxy)ethyl]propanamide),wherein for example, the linker is covalently attached to a FGF21peptide sequence through the thiol group of a cysteine in the FGF21peptide sequence (e.g., A129C) and the linker is conjugated to the Fabregion of the antibody. In some embodiments, two FGF21 peptide sequences(such as disclosed herein) are conjugated to a scaffold antibody (e.g.,as depicted in FIG. 12, e.g., a FGF21 homologue (e.g., a modifiedFGF21)-linker-Ab-linker-FGF21 homologue. In other embodiments a FGF21peptide sequence and an Exendin4 peptide sequence are conjugated to anantibody.

For example, provided herein is a long acting FGF21 comprising:

In other embodiments, long acting FGF21 forms may include: pegylatedFGF21 (e.g, that includes a FGFR21 with a R131AcF modification, coupledto PEG); modified FGF21 proteins with e.g., a FGF21 L118C, A1343C and/ora S167A modification, which has the effect, for example, to increasestability and/or limit or prevent glycosylation in yeast; and Fc-FGF21fusion constructs ((e.g, with FGF21 modifications L98R and/or P171G, forexample, a fusion protein having an IgG constant domain, a FGF21 proteinor modified protein and linker sequence associated with the IgG constantdomain FGF21 protein). Contemplated long acting FGF21 forms may includeFGF21 mimetics, such as FGFR1b/1c agonist antibodies; mimAB1agonistantibodies, and FGF1c/β-klothobispecific.

Exemplary wild type FGF21 human and mouse mRNA and protein sequencesinclude:

Human FGF21 gene, complete cds (GenBank Accession No. DQ847413;SEQ ID NO: 2) CACAGCACAGCAGGATGACTGCGGGCAGGCCTAGATAATACCCAGCCTCCCACAAGAAGCTGGTGGAGCAGAGTGTTCCCTGACTCCTCCAAGAAAAGGAGATTCCCTTTCGTGGTCTGCTGAGTAACGGGTGCCTTCCCAGACACTGGCGTTCCCGCTTGACCAAGGAGCCCTCAAGAGGCCCTTATGCCGGTGTGACAGAAGGCTCACCTCTTGCCTTCTAGGTCACTTCTCACAATGTCCCTTCAGTACCTGACCCTATACCCACCGGTTGTTTCCTGGTTATATTAGTTATACAACAAAGAATAAAAGTAATAGCTAATGATTAATAATGTTTACACTAATGATTGATACTGTCCATGATCATCTCTATATCTAATTTGTATGATAACTATTCTTATTCTAACTATTTTCTTTATTATACTGAAACAGTTTGTGCCTTCAGTCTCTTGCCTCGGCACCTGGGTAATCCTTCCCCACAGACTGACCCTCCCATTCAAGATACATCAATGTCAAAGACTCAGGAGTTTGACTTGATTCCCAGAAGTTTAACCATCATCTCCCCAGGCTCGGGACTCCCAGCACCCAGACCCTTCTGCTCACACCCAGCAGTCCAGGCCCCCAGACCCTCCTCCCTCAGACTTAGGAGTCCAGGCTCCCGGCCCCTCCTTCCTCAGACCCAGGAGTCCAAGCCCCCTGCCCCTCCTTCCTCAGACCCAGGAGTCCAGGACCCCAGCCCCTCCTTCCTCACACCCACGAGTCCAGATCCCTAGCCCCTACTCCCTCAGACCCAGGAGTCCAGACCAAAGCTCCCTCCTCCCTCAGACCCAGGAGCCCAAGTTCCCCAGCCCCTCCTCCCTCAGATCCAGGAGTACAGGCCCAGCCCCTCCTCCCTCAGACCCCTCCTCCCTCAGATCCAGGAGTACAGGCCCAGACCCTCCTCCCTCAGACCCAGGAGTCCAGGCCCCCCACCCCTCCTCCCTCAGACCCAGGAGTCCAGAGCCCCAGCCCTCCTCCCTCAGACACAGAAGGCCTACCCTTGCACCCTTAGGGGCTCCAGGAAATTAGCCAACCTGTCTTCCCTCTGGGTGCCCACTCCAGGGCCTGGCTTGGCTGCCAACTCCAGTCAGGGACTTTCAGCCACCCCTCCCCCCAGGTTATTTCAGGAGCACCTGCCTGGGCCTGGGATGGCTTCTCTGGTGAAAGAAACACCAGGATTGCATCAGGGAGGAGGAGGCTGGGATGTCCAGGGTCTGAGCATCTGAGCAGGGACAGATGAGGTTGAGGTTGGCCCACGGCCAGGTGAGAGGCTTCCAAGGCAGGATACTTGTGTCTCAGATGCGGTCGCTTCTTTCATACAGCAATTGCCGCCTTGCTGAGGATCAAGGAACCTCAGTGTCAGATCACGCCCTCCCCCCAAACTTAGAAATTCAGATGGGGCGCAGAAATTTCTCTTGTTCTGCGTGATCTGCATAGATGGTCCAAGAGGTGGTTTTTCCAGGAGCCCAGCACCCCTCCTCCCTCCGACTCAGGTGCTTGAGACCCCAGATCCTTCTCTCTGAGACTCAGGAATGTGGGCCCCCAGCCCCTTTCACCTGGGTCCCAGCTAACCCGATCCTCCCCTCCCTCATCCCCTAGACCCAGGAGTCTGGCCCTCCATTGAAAGGACCCCAGGTTACATCATCCATTCAGGCTGCCCTTGCCACGATGGAATTCTGTAGCTCCTGCCAAATGGGTCAAATATCATGGTTCAGGCGCAGGGAGGGTGATTGGGCGGGCCTGTCTGGGTATAAATTCTGGAGCTTCTGCATCTATCCCAAAAAACAAGGGTGTTCTGTCAGCTGAGGATCCAGCCGAAAGAGGAGCCAGGCACTCAGGCCACCTGAGTCTACTCACCTGGACAACTGGAATCTGGCACCAATTCTAAACCACTCAGCTTCTCCGAGCTCACACCCCGGAGATCACCTGAGGACCCGAGCCATTGATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGGCTGTGGGTTTCTGTGCTGGCTGGTCTTCTGCTGGGAGCCTGCCAGGCACACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTGAGTGTGGGCCAGAGCCTGGGTCTGAGGGAGGAGGGGCTGTGGGTCTGGATTCCTGGGTCTGAGGGAGGAGGGGCTGGGGGCCTTGGCCCCTGGGTCTGAGGGAGGAGGGGCTGGGGATCTGGACTCCTGGGTCTGAGGGAGGAGGGGCTGGGGATCTGGGCCCCTGGGTCTGAGGGAGGAGGGGCTGGGTCTGGACCCCTGGGTCTGAGGGAGGAGGGGCTGGGGGTCTGGACTCTTGGGTTTGAAGAAGGAAGGGCTGGGGTCCTGGACTCTTGGGTCTGAGTTGGGAGGGGGCTTTGGCTTGGGCTTCTCCTGGGTCTGAGGGAGGAGGTAGGCTGTGGGCTTGGACTCCCAGGGCTGGGACAGAGCCGGATGGTGGGACAGAGTCGGGTGGTGGGACAGTCCCGGGTGGGAGAGGTCCTCGAACCACCTTATCGCTTTCACCCCTTAGGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGGTGAGTTTCCAGGACCCTCCTCACCACCCACCATGCTCCTCCTATATGTCGCCCTCACAGCCTGGGGTGCCTTGTCTTGCTCATCCCCCCCGGAGCCAGACTTGATTCTATTTGCTCTGCACGCCCCCAGCTGCAACATTTGGAGGTTGAAGTTGTCATCAGTGTTTGCAAGATGAGGAAACTGAGGCCCAGGCCGGGGCGCCAGTGACCTCAATCATGTGATGTGTGGATGCTGGAGCGGCCTGAGGCTCAGGTTATTGGGAGTCTCGTGATTCAGTAACCCCTGCTCCTGCCCACACGGCCCCTGTGTGCACGGCTCATGCTGGGCACAGGGACACTCGGGGAAGCCATGGCCAGTAAAGTGACCAGGACCTTGAGTGCTAGGGAGACACCCCG CCTGGCCTGAGAGAGCACTGATGGCTCCGAGGGCTGGAATGTTCTCTGTGAAGTCTGAACTGGGAGGCAGGTCCCTGCAGGAGAGCCCTGGGGTAAAAAACAAAACCTGCCTTGCTGTTTTGTTTCCTAGAGGAGGGGCTGGGGGCCTGGACTCCTGGGTCTGAGGGAGGAGGGGCTGGGGGCCTGGACCCCTGGGTCTGAGGGAGGAGGGGCTGGGGGCCTGGAACCCCGGGTCTGAGGGAGGAGAGGCTGGGGCCTGGAACCCCGGGTCTGAGGGAGGAGAGGCTGGGGCCTGGAACCCCGGGTCTGAGGGAGGAGGCGCTGGGGGCCTGGACTCCTGGGTCGGATGGAGGAGAAACTAGGGTCTGGACCCCTGGGTCTGAGGGAGGAGGCGCTGGGGGCCTGGACCCCTGGGTCTGAGGGAGGCAGGGCTGGGGCCTGGATCCTGGGTCTTACATCAGGAAAACAGAGGAACCCTGTCTCTGATCCTGTTTTTGTCCCCTAGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACCCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCAGCTACGCTTCCTGAAGCCAGAGGCTGTTTACTATGACATCTCCTCTTTATTTATTAGGTTATTTATCTTATTTATTTTTTTATTTTTCTTACTTGAGATAATAAAGAGTTCCAGAGGAGGATAAGAATGAGCATGTGTGAGTGTCTGAGGGAAGACATGGCAGCTGTTTTGTCTCCCTTGGCCCGGACAATCCCCTCTACACCTCCCCTCACGTGGTCCGAGGGTCCTGGCTTCCCACTGGGCCTCACTTTTTTCTTTTCTTTTCTTTTTTTTTTTTTGAGACGGAGTCTCGCTCTGTCACCCAGGCTGGAGTGCAGTGGCGCGATCTTGGCTCACTCCAACCTCCGCCTCCCAGGTTCAAGCAATTCTCCTGCCTCAGCCACCCGAGTAGCTGTGATTACAGGCGTGCGCCACCACACCCAGCTAATTTTGTAATTTTAGTAGAGACAGGGTTTCGCCATGTTGGCCAGGATGCTCTCCATCTCTTGACTTCATGACCTGCCTGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCTTGAGTCACTGTGCCCAGCCCAGCCTCACTTTTCTACTCTGCTAAAGTGTCCCCAGGGACTGTGGACTATCCCTGCTCTCTGAAAGGACAAGACTGGCCGGGAGTGGTGGCTTACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGTGGATCACGAGGTCAGGAGATTGAGACTATCCTGGCTAATACGATGAAACCCCGTCTCTACTAAAAATACAAAAACAAAATTAGCTGGGCGTGGTGGCGGGCGCCTGTAGTCCCAGCTACTCCGGAGGCTGAGGCAGAATGGCGTGAATGCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCGCGCCACTGCACTCCAGCCCAGGCCACAGAGCGAGATTCCATCTCAAAAAAATAAATAAATAAATAAATAAATAAATAAATATAAAAATAAAATGAAAGAGCAGGACTTCTTTCTACAACCCCTCAACTTGTGTGAGCGTTGTGTAACTATTTCATAGAGCTACCTCAATAACAGGGGAGCTTTTACGAGGTGACACAGCACACTCACATCCTCATGGGAGATGTAGTTTTCTGGCATCATTTAGCAGCAGGAATGAGATCTGTTGGGCCTCAAATCTGGGACAAGGACTCCTGGGTCCTGGAGTAGGTTTGGGGCTAGTGTAACACCCAAGTTCTGGGGAATCAGTGGGCTGGACATCTGGACACCTGGATCACAGGAGAACTGGGGACTGCAGACTTAGGCATCCTGGTCTGAGAAAAAAGGGGCTGGAGGGTGGGAGTTTGGGTTCTCAGGAAAAGGAGCTGAAACCTGGAATTCTTCCATCTGGGTCCTTATGAAC mRNA:Human FGF21 mRNA, complete cds (GenBank Accession NM_019113;SEQ ID NO: 3) GAGGCTTCCAAGGCAGGATACTTGTGTCTCAGATGCGGTCGCTTCTTTCATACAGCAATTGCCGCCTTGCTGAGGATCAAGGAACCTCAGTGTCAGATCACGCCCTCCCCCCAAACTTAGAAATTCAGATGGGGCGCAGAAATTTCTCTTGTTCTGCGTGATCTGCATAGATGGTCCAAGAGGTGGTTTTTCCAGGAGCCCAGCACCCCTCCTCCCTCCGACTCAGACCCAGGAGTCTGGCCCTCCATTGAAAGGACCCCAGGTTACATCATCCATTCAGGCTGCCCTTGCCACGATGGAATTCTGTAGCTCCTGCCAAATGGGTCAAATATCATGGTTCAGGCGCAGGGAGGGTGATTGGGCGGGCCTGTCTGGGTATAAATTCTGGAGCTTCTGCATCTATCCCAAAAAACAAGGGTGTTCTGTCAGCTGAGGATCCAGCCGAAAGAGGAGCCAGGCACTCAGGCCACCTGAGTCTACTCACCTGGACAACTGGAATCTGGCACCAATTCTAAACCACTCAGCTTCTCCGAGCTCACACCCCGGAGATCACCTGAGGACCCGAGCCATTGATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTGTGGGTTTCTGTGCTGGCTGGTCTTCTGCTGGGAGCCTGCCAGGCACACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACTCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCACTACGCTTCCTGAAGCCAGAGGCTGTTTACTATGACATCTCCTCTTTATTTATTAGGTTATTTATCTTATTTATTTTTTTATTTTTCTTACTTGAGATAATAAAGAGTTCCAGAGGAGGATAAAAAAAAAAAAAAAAAAAAAAA mRNA:Mus musculas FGF21, mRNA (GenBank Accession No. NM_020013; SEQ ID NO: 4)AGACAGCCTTAGTGTCTTCTCAGCTGGGGATTCAACACAGGAGAAACAGCCATTCACTTTGCCTGAGCCCCAGTCTGAACCTGACCCATCCCTGCTGGGCACCGGAGTCAGAACACAATTCCAGCTGCCTTGGCTCCTCAGCCGCTCGCTTGCCAGGGGCTCTCCCGAACGGAGCGCAGCCCTGATGGAATGGATGAGATCTAGAGTTGGGACCCTGGGACTGTGGGTCCGACTGCTGCTGGCTGTCTTCCTGCTGGGGGTCTACCAAGCATACCCCATCCCTGACTCCAGCCCCCTCCTCCAGTTTGGGGGTCAAGTCCGGCAGAGGTACCTCTACACAGATGACGACCAAGACACTGAAGCCCACCTGGAGATCAGGGAGGATGGAACAGTGGTAGGCGCAGCACACCGCAGTCCAGAAAGTCTCCTGGAGCTCAAAGCCTTGAAGCCAGGGGTCATTCAAATCCTGGGTGTCAAAGCCTCTAGGTTTCTTTGCCAACAGCCAGATGGAGCTCTCTATGGATCGCCTCACTTTGATCCTGAGGCCTGCAGCTTCAGAGAACTGCTGCTGGAGGACGGTTACAATGTGTACCAGTCTGAAGCCCATGGCCTGCCCCTGCGTCTGCCTCAGAAGGACTCCCCAAACCAGGATGCAACATCCTGGGGACCTGTGCGCTTCCTGCCCATGCCAGGCCTGCTCCACGAGCCCCAAGACCAAGCAGGATTCCTGCCCCCAGAGCCCCCAGATGTGGGCTCCTCTGACCCCCTGAGCATGGTAGAGCCTTTACAGGGCCGAAGCCCCAGCTATGCGTCCTGACTCTTCCTGAATCTAGGGCTGTTTCTTTTTGGGTTTCCACTTATTTATTACGGGTATTTATCTTATTTATTTATTTTAGTTTTTTTTTCTTACTTGGAATAATAAAGAGTCTGAAAGAAAAATGTGTGTTMus musculas FGF21 protein (AAH49592) (SEQ ID NO: 5)MEWMRSRVGTLGLWVRLLLAVFLLGVYQAYPIPDSSPLLQFGGQVRQRYLYTDDDQDTEAHLEIREDGTVVGAAHRSPESLLELKALKPGVIQILGVKASRFLCQQPDGALYGSPHFDPEACSFRELLLEDGYNVYQSEAHGLPLRLPQKDSPNQDATSWGPVRFLPMPGLLHEPQDQAGFLPPEPPDVGSSDPLSMVEPLQGRSPSYASHuman FGF21 protein (GenBank accession AAQ89444) (SEQ ID NO: 6)MDSDETGFEHSGLWVSVLAGLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS

Beta Klotho

Beta klotho contributes to the transcriptional repression of cholesterol7-alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acidsynthesis. Beta klotho increases the ability of FGFR1 and FGFR4 to bindFGF21. Beta klotho acts as a co-receptor for FGF21. The fibroblastgrowth factor (FGF) 19 subfamily of ligands, FGF19, FGF21, and FGF23,function as hormones that regulate bile acid, fatty acid, glucose, andphosphate metabolism in target organs through activating FGF receptors(FGFR1-4). Klotho and 1 Klotho, homologous single-pass transmembraneproteins that bind to FGFRs, are required for metabolic activity ofFGF23 and FGF21, respectively. Like FGF21, FGF19 also requires βKlotho.Both FGF19 and FGF21 can signal through FGFR1-3 bound by βKlotho andincrease glucose uptake in adipocytes expressing FGFR1. Additionally,both FGF19 and FGF21 bind to the βKlotho-FGFR4 complex; however, onlyFGF19 signals efficiently through FGFR4. Accordingly, FGF19, but notFGF21, activates FGF signaling in hepatocytes that primarily expressFGFR4 and reduces transcription of CYP7A1 that encodes the rate-limitingenzyme for bile acid synthesis. The expression of βKlotho, incombination with particular FGFR isoforms, determines thetissue-specific metabolic activities of FGF19 and FGF21.

Exemplary Beta Klotho human and mouse mRNA and protein sequencesinclude:

Human Beta Klotho gene, Cyp7a1 gene, linear DNA (GenBank AccessionNo. DD362478) (SEQ ID NO: 7)ACCCTGGGCCTGGCCCAAGAAACTATACATTCCTCCTGGGAATCTGGGGCTGTGATGGGAGGGGTTGCCATGAAGACTTCTGACATGCCCTGGAGACATTTTCCCCATGGTCTTGGGGATTAACATTCAGCTCCTTGTTACTTATGCAAATTTCTGCAGCTGGCTTGAATTTCTCCTCAGAAAATGAGATTTTCTTTTCTATCGCATTGTCAGGCTGCAAAT TTTCCAAACTTTTGTGCTCTGCTTCCCTTATAAAACTGAAGGCCTGGCCAGGTGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGGAGCTGAGACGGGCGAATCACGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCTGTCTCTACTAAAAATACAAAAAGTTAGCTGGGCATAGTCATGGGTGCCTTTAATCCCAGCTACTTGGGAGACTGAGGCAGGAGAATCGTTTGAATCCAGGAGGCGGAGGTTGCAGTGAGTCGAGATCACACCACTGCACTCCAGCCCTGGTAACATAGTAAGACTCTGTCTCAAAAAAAAAAAAAAAAAAACAAAACTGAATGCCTTTAACAACACCCAAGTTGCTTCTTGAATGCTTTGCTGCTTAGAAATTTCTTCTGCCAGATACCCTAAATCATCTCTCTTAGGTTCCAAGTTCCACAAATCTCTAGGGCAGGGACAAAACGCTGCCAGTCTCTTTACTAAAACATAACAAGAGTCACCCTTGCTCTAGTTCCCAAAAAGTTCCTCATCTCCATCTGAAACCACCCCCGCCTAGATTTCGTTGTCCATATCATTATCAGCATTTTGGTCAAAGTCATTCAACAGGCCTCTAGGGAGTTCCAAACTTGCTCACATTCTCCTGTCTTCTTCTGAGCCCTCCAAACTGTTCCAATCCCTACCTGTTACCCAGTTCCAAAGTCGCTTCCACATTTTTGGTTATCTTTTCAGCCGTGCCCCACTCTACTGGTACCAGTTTACTGTATTAGTCGATTTTCATGCTGCTGATAAAGACATACCTGAAACTGGACAATTTACAAAAGAAAGAGGTTTATTGGACTTACAATTCTACATCACTTGGGAGGCCTCACAATCATGATGGAAGGAGAAAGGCACATCTCACATGGCAGCAGACAAGAAAAGAGCTTGTGCAGGGAAACTCCTCTTTTTAAAACCATCAGATCTCATGAAATTTATTCATTATCATGACAATAGCACAGGAAAGAACTGCACCCATAATTCAGTCACCTCCTACCAGGTTCCTCCCACAACACGTGAGAATTCAAGATGAGATTTGGATGGGGACACAGCCAAACCATGTCACACTACCATGCCTGACTTCCTTTCCATTTTTGTATATTTGCTTGTTCTTCATTTGCCCGAGAAGTAACTCTAAAGGGCTGTATTATTTGGATATTAGATTGGCATTTTATCTGACTGGGATATCTTGCTGTGATTGTCCATGTATAAGATCAGCTTTTCTATAAACCATATTTTTAAAAAGATATATTAATTTTTTAAAAATCCACCTGTCTAAATAAATGCACAAAGCCCCCCAAAAACCTAGATTCTAAGAAAAATCTATGTACTGCCATACAATGATTGATATTAATATTTATGGTGATAAATTACACACAAAAAATGTGTGATCTCTGTTTAAACAGGCAAAAACAAAAAACACATGAAATAAATCTATGGCATCTATAGCCAAAACTGGAAACAACCCACATATCCATCAATAGGAAATCAGTTAAATAAATTATAGTACATTTATCCAATGGAAGATTAAGCACATATTCAATATAATTATTTATACACACATATAGATACACACATGTATAAATATAGAGAATACTGTGGGTGTATGTGTGTGTGTGTTTATATACATATATATACACACACAGTACTGTTGCCTACCTTCTTTTGTCTTAATTCTGTGAACTCTCATTCACTCTGCTTCAGTAGGATACATCCTTCTTTTTGGTTCTTAGACTCACCAAGTTGATCCTTGACTCAAGACATTGCATTTGCTGCTTCCTCTTCCTGGAATATCCTTCCTTCTGATATTCACATGAGTAGTCTCTTCTTGTCATTCAGATCTCAAATGTCACAATTTCAGAGAGCCCATCTCTGATCATCATATCTAAAGTTGTCCTCATTCCCCCATAGCTTTCTATACCATGTTTTATTTTTTTCATAACATGTATTTTATTACTCCTTTCTCCATTGGAATAGAATCTCCATTAGATTAGGAAATCTGCCTATCTTATTAATGCCTGCAACTGGAATACTTTTGAAGAGTTCTTGGCACGTAATAAATACTCAACTAATATTTTTGTGTACACAGAAATAAAGTTTGGAAGAACAGATGCCAAATTGTTACTAGTGGTTACTTCTGAGTAAAGGAGTAGCATGGTAGGTAAATTATTAATAGATGTTCACTTTCCACCAAGATATGTTTTAGTTAGTCTTAACTTACTTGAAATGAAATTTATTACTTTAATAATTAGAAACATTGATAAACATTTTAGTCACAAGAATGATAGATAAAATTTTGATGCTTCCAATAAGTTATATTTATCTAGAGGATGCACTTATGTAGAATACTCTCTTGAGGATGTTAGGTGAGTAACATGTTACTATATGTAGTAAAATATCTATGATTTTATAAAAGCACTGAAACATGAAGCAGCAGAAACGTTTTTCCCAGTTCTCTTTCCTCTGAACTTGATCACCGTCTCTCTGGCAAAGCACCTAAATTAATTCTTCTTTAAAAGTTAACAAGACCAAATTATAAGCTTGATGAATAACTCATTCTTATCTTTCTTTAAATGATTATAGTTTATGTATTTATTAGCTATGCCCATCTTAAACAGGTTTATTTGTTCTTTTTACACATACCAAACTCTTAATATTAGCTGTTGTCCCCAGGTCCGAATGTTAAGTCAACATATATTTGAGAGAACTTCAACTTATCAAGTATTGCAGGTCTCTGATTGCTTTGGAACCACTTCTGATACCTGTGGACTTAGTTCAAGGCCAGTTACTACCACTTTTTTTTTTCTA ATAGAATGAACAAATGGCTAATTGTTTGCTTTGTCAACCAAGCTCAAGTTAATGGATCTGGATACTATGTATATAAAAAGCCTAGCTTGAGTCTCTTTTCAGTGGCATCCTTCCCTTTCTAATCAGAGATTTTCTTCCTCAGAGATTTTGGCCTAGATTTGCAAAHomo sapiens, klotho beta (KLB) mRNA (GenBank Accession No. NM_175737)(SEQ ID NO: 8) ATCCTCAGTCTCCCAGTTCAAGCTAATCATTGACAGAGCTTTACAATCACAAGCTTTTACTGAAGCTTTGATAAGACAGTCCAGCAGTTGGTGGCAAATGAAGCCAGGCTGTGCGGCAGGATCTCCAGGGAATGAATGGATTTTCTTCAGCACTGATGAAATAACCACACGCTATAGGAATACAATGTCCAACGGGGGATTGCAAAGATCTGTCATCCTGTCAGCACTTATTCTGCTACGAGCTGTTACTGGATTCTCTGGAGATGGAAGAGCTATATGGTCTAAAAATCCTAATTTTACTCCGGTAAATGAAAGTCAGCTGTTTCTCTATGACACTTTCCCTAAAAACTTTTTCTGGGGTATTGGGACTGGAGCATTGCAAGTGGAAGGGAGTTGGAAGAAGGATGGAAAAGGACCTTCTATATGGGATCATTTCATCCACACACACCTTAAAAATGTCAGCAGCACGAATGGTTCCAGTGACAGTTATATTTTTCTGGAAAAAGACTTATCAGCCCTGGATTTTATAGGAGTTTCTTTTTATCAATTTTCAATTTCCTGGCCAAGGCTTTTCCCCGATGGAATAGTAACAGTTGCCAACGCAAAAGGTCTGCAGTACTACAGTACTCTTCTGGACGCTCTAGTGCTTAGAAACATTGAACCTATAGTTACTTTATACCACTGGGATTTGCCTTTGGCACTACAAGAAAAATATGGGGGGTGGAAAAATGATACCATAATAGATATCTTCAATGACTATGCCACATACTGTTTCCAGATGTTTGGGGACCGTGTCAAATATTGGATTACAATTCACAACCCATATCTAGTGGCTTGGCATGGGTATGGGACAGGTATGCATGCCCCTGGAGAGAAGGGAAATTTAGCAGCTGTCTACACTGTG GGACACAACTTGATCAAGGCTCACTCGAAAGTTTGGCATAACTACAACACACATTTCCGCCCACATCAGAAGGGTTGGTTATCGATCACGTTGGGATCTCATTGGATCGAGCCAAACCGGTCGGAAAACACGATGGATATATTCAAATGTCAACAATCCATGGTTTCTGTGCTTGGATGGTTTGCCAACCCTATCCATGGGGATGGCGACTATCCAGAGGGGATGAGAAAGAAGTTGTTCTCCGTTCTACCCATTTTCTCTGAAGCAGAGAAGCATGAGATGAGAGGCACAGCTGATTTCTTTGCCTTTTCTTTTGGACCCAACAACTTCAAGCCCCTAAACACCATGGCTAAAATGGGACAAAATGTTTCACTTAATTTAAGAGAAGCGCTGAACTGGATTAAACTGGAATACAACAACCCTCGAATCTTGATTGCTGAGAATGGCTGGTTCACAGACAGTCGTGTGAAAACAGAAGACACCACGGCCATCTACATGATGAAGAATTTCCTCAGCCAGGTGCTTCAAGCAATAAGGTTAGATGAAATACGAGTGTTTGGTTATACTGCCTGGTCTCTCCTGGATGGCTTTGAATGGCAGGATGCTTACACCATCCGCCGAGGATTATTTTATGTGGATTTTAACAGTAAACAGAAAGAGCGGAAACCTAAGTCTTCAGCACACTACTACAAACAGATCATACGAGAAAATGGTTTTTCTTTAAAAGAGTCCACGCCAGATGTGCAGGGCCAGTTTCCCTGTGACTTCTCCTGGGGTGTCACTGAATCTGTTCTTAAGCCCGAGTCTGTGGCTTCGTCCCCACAGTTCAGCGATCCTCATCTGTACGTGTGGAACGCCACTGGCAACAGACTGTTGCACCGAGTGGAAGGGGTGAGGCTGAAAACACGACCCGCTCAATGCACAGATTTTGTAAACATCAAAAAACAACTTGAGATGTTGGCAAGAATGAAAGTCACCCACTACCGGTTTGCTCTGGATTGGGCCTCGGTCCTTCCCACTGGCAACCTGTCCGCGGTGAACCGACAGGCCCTGAGGTACTACAGGTGCGTGGTCAGTGAGGGGCTGAAGCTTGGCATCTCCGCGATGGTCACCCTGTATTATCCGACCCACGCCCACCTAGGCCTCCCCGAGCCTCTGTTGCATGCCGACGGGTGGCTGAACCCATCGACGGCCGAGGCCTTCCAGGCCTACGCTGGGCTGTGCTTCCAGGAGCTGGGGGACCTGGTGAAGCTCTGGATCACCATCAACGAGCCTAACCGGCTAAGTGACATCTACAACCGCTCTGGCAACGACACCTACGGGGCGGCGCACAACCTGCTGGTGGCCCACGCCCTGGCCTGGCGCCTCTACGACCGGCAGTTCAGGCCCTCACAGCGCGGGGCCGTGTCGCTGTCGCTGCACGCGGACTGGGCGGAACCCGCCAACCCCTATGCTGACTCGCACTGGAGGGCGGCCGAGCGCTTCCTGCAGTTCGAGATCGCCTGGTTCGCCGAGCCGCTCTTCAAGACC GGGGACTACCCCGCGGCCATGAGGGAATACATTGCCTCCAAGCACCGACGGGGGCTTTCCAGCTCGGCCCTGCCGCGCCTCACCGAGGCCGAAAGGAGGCTGCTCAAGGGCACGGTCGACTTCTGCGCGCTCAACCACTTCACCACTAGGTTCGTGATGCACGAGCAGCTGGCCGGCAGCCGCTACGACTCGGACAGGGACATCCAGTTTCTGCAGGACATCACCCGCCTGAGCTCCCCCACGCGCCTGGCTGTGATTCCCTGGGGGGTGCGCAAGCTGCTGCGGTGGGTCCGGAGGAACTACGGCGACATGGACATTTACATCACCGCCAGTGGCATCGACGACCAGGCTCTGGAGGATGACCGGCTCCGGAAGTACTACCTAGGGAAGTACCTTCAGGAGGTGCTGAAAGCATACCTGATTGATAAAGTCAGAATCAA AGGCTATTATGCATTCAAACTGGCTGAAGAGAAATCTAAACCCAGATTTGGATTCTTCACATCTGATTTTAAAGCTAAATCCTCAATACAATTTTACAACAAAGTGATCAGCAGCAGGGGCTTCCCTTTTGAGAACAGTAGTTCTAGATGCAGTCAGACCCAAGAAAATACAGAGTGCA CTGTCTGCTTATTCCTTGTGCAGAAGAAACCACTGATATTCCTGGGTTGTTGCTTCTTCTCCACCCTGGTTCTACTCTTATCAATTGCCATTTTTCAAAGGCAGAAGAGAAGAAAGTTTTGGAAAGCAAAAAACTTACAACACATACCATTAAAGAAAGGCAAGAGAGTTGT TAGCTAAACTGATCTGTCTGCATGATAGACAGTTTAAAAATTCATCCCAGTTCCATATGCTGGTAACTTACAGGAGATATACCTGTATTATAGAAAGACAATCTGAGATACAGCTGTAACCAAGGTGATGACAATTGTCTCTGCTGTGTGGTTCAAAGAACATTCCCTTA GGTGTTGACATCAGTGAACTCAGTTCTTGGATGTAAACATAAAGGCTTCATCCTGACAGTAAGCTATGAGGATTACATGCTACATTGCTTCTTAAAGTTTCATCAACTGTATTCCATCATTCTGCTTTAGCTTTCATCTCTACCAATAGCTACTTGTGGTACAATAAATTATTTTTAAGAAGTAAAACTCTGGGGCTGGACGCTGTGGCTCACACCTGTAATCTCAG CACTTTGGGAGGCCGAGGCGGGGAGATCACCTGAGGTGAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCATGTCTCTACTAAAAATACAAAAAATTAGCCAGGCGTGGTGACAGTGGCACCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGAAGTTTGAACCCAGGAAACAGGTTACAGTAGGCCAAAATTGCGCCACTGCACTCCAGCCTAGGCGACAACAGCAAGACTGTGTCCAAAAAAAAAAAAAAAAGCAAAAGCAAAACTTTGTTTTGTTAGACTCTACAGCAGAGATTTAACACCCTTCTTTAAACTGGGTAGTCAGTGATAGATAATATATATTCTGTCACTTCTAATAAGGTGCCTTCTCCTTTAGGTCAGGGTGGTTCTAAAATGGAAAGAAAACACAATAGGGTAAGTAGTGCTTGTCTAAG CCAGTTACAACACAGACTCTTAAAGAGGATCAAGCCCTTCATTTTTCTAACAACAAAAAATCACCTATAGAATATCTAATTTGTGATCTTTTACTAGATCTGATTTTTTAAAATAATGTAATTTCCGGCCAGGCACGGTGGCACCGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCAGGTGGATCACCTGAGGTTAGGAGTTCGAGACTAGCCTGGCCAACATGGCAAAACCCCATCTCTACTAAAAATACAAAAGTTAGCCGGGCATGGTGGTGGGCACCTGTAATCCCAGCTACTCAGGAGGCCGAGGCAGGAGAATCGCTTGAACCCGAGAGGCAGAGGTTGCAATGAGCCAAGATCGTGCCATTGCACTCCAGCCTGGGGGACAGGGCAAGACTGTCTCTCAAAATAAAAAAAAATAATAAAAATAAAAATAAAAGTAATTTCCA AAACCTCATCTCATGGAAAGATCACAGGATGAAGGAAAGCTAGACTCAACTCTGTGAATAGAAGTTGCTATACTGTAAGTAAAGCAACAATTCAGAATACTGAATGAGTTTAAATTGTTTTATATAGCACCCTTTTGGGCTAGGGTTAATTACTAGATCTGACTTGGATAATTTGACACT TTGGGAAATGAACTCTGTTCTTGAGACTTGTTCAGTGTATTTTAAACATCTGAGGAAGAAAACTTAAATATGCACCTATTTATACCTATTCTTTCTTTAGGTCAACATTTAACACCCACTGCATACATTAATTTGTCCTTGTCTGCTCACTCCAGCAATTTAGACCTTAACAGTCACAAGAGACGTTCTTCTGTTACAAAGCCTTAGTAAATTAAGGCAGTTTTGATT ATATTCTAGGTCCACCTATGTCTGAAGCTAAATTCAGTATCTAACTGCTAATGAACAAGTTTCCAAAATACTGTAAAAATACAATTAGTCAATTTGAGTAAATGCAAATATGATGAGAAATCAATTTGCTATTTGGCCTGGCAAATGGGAACAGTAAAATTCTGCTTTACTCTTCTCTAGTCTCCTTGCCCCAGCTGCACCCACTACCCCAAAGTTGGCAGTTTTGAGGTATGATTTTCAAGGAATTTTTTTAGTATTAACATCTCCCTCTGAGAACTATGTACCTAAGGTCACGCATACAACTAGTCAATTCTGTTTTTATTACTCTAACTATGTAGAAACAGTAAGTCACTTAAAACAATCACTTGGCTGGGTTTTTTCCCCTTTGTGCCACATTGATTCACCCTGACCCAAGAACTCCAGGGAAAATTCTTTAATGTCAACTGGGCAACTCATTAACCTCTCTTTAACATCAAGGGCTTGGGAAAAAAAAAAAAAAGGTTAGCCACAGGAATAACAAAAACCTGGAATTTATCTTTCAGGTTTTGCTTTCTCTTTCTCACTTTGTTTAAAGTATCTCGTACTCACAGTTCACAAATTAACCTTCACTGTCTCTTTCACATTAAGAGCTTATGCTTAAAGCATGCCCCCCTTTTCTAACTTGCTGGTTTACCATAAACTCCCCTAAGTAATAAAATTCCTAACCCAGTACTGAGAGTCCTCCTTCTCTGCCACTTGGGCATTATTTTACTAGTTTTTAAGCCATCATCGCACAAGAATCCAAAAACCCTTAAATTTTTTAACCACTGGCAAATATGTACAGCAAATTAGGTTAAGCATTTAATCTGGCTCATGCTCTATCATACTAAATATTCAGGTTTATCATAAACTCCTTAAAAACCATCAAAGGTCAACCAGAAACTGATAACTCTTGAAAGGAGCAAACAGGTAAGATCTTTGGAGTTTAAGCTTTTCTGAGATGTGTTGTGAAAAATCTAACGTGTTTATCGTATATTCAATGTAACAACCTGGAGAATCACAACTATATTTAAAGAGCCTCTGGAAAATGAGGCCAGTACAGTG TGACTACATGTTTAATTTTCAATGTAATTTATTCCAAATAAACTGGTTCATGCTGACCACTTGTATTCAAC TAAHuman Beta Klotho (GenBank Accession No. NP_783864) (SEQ ID NO: 9)MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDRQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKR VVSMus musculas beta klotho (KLB), mRNA (GenBank Accession No. NM_031180)(SEQ ID NO: 10) AATGAAGACAGGCTGTGCAGCAGGGTCTCCGGGGAATGAATGGATTTTCTTCAGCTCTGATGAAAGAAACACACGCTCTAGGAAAACAATGTCCAACAGGGCACTGCAAAGATCTGCCGTGCTGTCTGCGTTTGTTCTGCTGCGAGCTGTTACCGGCTTCTCCGGAGACGGGAAAGCAATATGGGATAAAAAACAGTACGTGAGTCCGGTAAACCCAAGTCAGCTGTTCCTCTATGACACTTTCCCTAAAAACTTTTCCTGGGGCGTTGGGACCGGAGCATTTCAAGTGGAAGGGAGTTGGAAGACAGATGGAAGAGGACCCTCGATCTGGGATCGGTACGTCTACTCACACCTGAGAGGTGTCAACGGCACAGACAGATCCACTGACAGTTACATCTTTCTGGAAAAAGACTTGTTGGCTCTGGATTTTTTAGGAGTTTCTTTTTATCAGTTCTCAATCTCCTGGCCACGGTTGTTTCCCAATGGAACAGTAGCAGCAGTGAATGCGCAAGGTCTCCGGTACTACCGTGCACTTCTGGACTCGCTGGTACTTAGGAATATCGAGCCCATTGTTACCTTGTACCATTGGGATTTGCCTCTGACGCTCCAGGAAGAATATGGGGGCTGGAAAAATGCAACTATGATAGATCTCTTCAACGACTATGCCACATACTGCTTCCAGACCTTTGGAGACCGTGTCAAATATTGGATTACAATTCACAACCCTTACCTTGTTGCTTGGCATGGGTTTGGCACAGGTATGCATGCACCAGGAGAGAAGGGAAATTTAACAGCTGTCTACACTGTGGGACACAACCTGATCAAGGCACATTCGAAAGTGTGGCATAACTACGACAAAAACTTCCGCCCTCATCAGAAGGGTTGGCTCTCCATCACCTTGGGGTCCCATTGGATAGAGCCAAACAGAACAGACAACATGGAGGACGTGATCAACTGCCAGCACTCCATGTCCTCTGTGCTTGGATGGTTCGCCAACCCCATCCACGGGGACGGCGACTACCCTGAGTTCATGAAGACGGGCGCCATGATCCCCGAGTTCTCTGAGGCAGAGAAGGAGGAGGTGAGGGGCACGGCTGATTTCTTTGCCTTTTCCTTCGGGCCCAACAACTTCAGGCCCTCAAACACCGTGGTGAAAATGGGACAAAATGTATCACTCAACTTAAGGCAGGTGCTGAACTGGATTAAACTGGAATACGATGACCCTCAAATCTTGATTTCGGAGAACGGCTGGTTCACAGATAGCTATATAAAGACAGAGGACACCACGGCCATCTACATGATGAAGAATTTCCTAAACCAGGTTCTTCAAGCAATAAAATTTGATGAAATCCGCGTGTTTGGTTATACGGCCTGGACTCTCCTGGATGGCTTTGAGTGGCAGGATGCCTATACGACCCGACGAGGGCTGTTTTATGTGGACTTTAACAGTGAGCAGAAAGAGAGGAAACCCAAGTCCTCGGCTCATTACTACAAGCAGATCATACAAGACAACGGCTTCCCTTTGAAAGAGTCCACGCCAGACATGAAGGGTCGGTTCCCCTGTGATTTCTCTTGGGGAGTCACTGAGTCTGTTCTTAAGCCCGAGTTTACGGTCTCCTCCCCGCAGTTTACCGATCCTCACCTGTATGTGTGGAATGTCACTGGCAACAGATTGCTCTACCGAGTGGAAGGGGTAAGGCTGAAAACAAGACCATCCCAGTGCACAGATTATGTGAGCATCAAAAAACGAGTTGAAATGTTGGCAAAAATGAAAGTCACCCACTACCAGTTTGCTCTGGACTGGACCTCTATCCTTCCCACTGGCAATCTGTCCAAAGTTAACAGACAAGTGTTAAGGTACTATAGGTGTGTGGTGAGCGAAGGACTGAAGCTGGGCGTCTTCCCCATGGTGACGTTGTACCACCCAACCCACTCCCATCTCGGCCTCCCCCTGCCACTTCTGAGCAGTGGGGGGTGGCTAAACATGAACACAGCCAAGGCCTTCCAGGACTACGCTGAGCTGTGCTTCCGGGAGTTGGGGGACTTGGTGAAGCTCTGGATCACCATCAATGAGCCTAACAGGCTGAGTGACATGTACAACCGCACGAGTAATGACACCTACCGTGCAGCCCACAACCTGATGATCGCCCATGCCCAGGTCTGGCACCTCTATGATAGGCAGTATAGGCCGGTCCAGCATGGGGCTGTGTCGCTGTCCTTACATTGCGACTGGGCAGAACCTGCCAACCCCTTTGTGGATTCACACTGGAAGGCAGCCGAGCGCTTCCTCCAGTTTGAGATCGCCTGGTTTGCAGATCCGCTCTTCAAGACTGGCGACTATCCATCGGTTATGAAGGAATACATCGCCTCCAAGAACCAGCGAGGGCTGTCTAGCTCAGTCCTGCCGCGCTTCACCGCGAAGGAGAGCAGGCTGGTGAAGGGTACCGTCGACTTCTACGCACTGAACCACTTCACTACGAGGTTCGTGATACACAAGCAGCTGAACACCAACCGCTCAGTTGCAGACAGGGACGTCCAGTTCCTGCAGGACATCACCCGCCTAAGCTCGCCCAGCCGCCTGGCTGTAACACCCTGGGGAGTGCGCAAGCTCCTTGCGTGGATCCGGAGGAACTACAGAGACAGGGATATCTACATCACAGCCAATGGCATCGATGACCTGGCTCTAGAGGATGATCAGATCCGAAAGTACTACTTGGAGAAGTATGTCCAGGAGGCTCTGAAAGCATATCTCATTGACAAGGTCAAAATCAAAGGCTACTATGCATTCAAACTGACTGAAGAGAAATCTAAGCCTAGATTTGGATTTTTCACCTCTGACTTCAGAGCTAAGTCCTCTGTCCAGTTTTACAGCAAGCTGATCAGCAGCAGTGGCCTCCCCGCTGAGAACAGAAGTCCTGCGTGTGGTCAGCCTGCGGAAGACACAGACTGCACCATTTGCTCATTTCTCGTGGAGAAGAAACCACTCATCTTCTTCGGTTGCTGCTTCATCTCCACTCTGGCTGTACTGCTATCCATCACCGTTTTTCATCATCAAAAGAGAAGAAAATTCCAGAAAGCAAGGAACTTACAAAATATACCATTGAAGAAAGGCCACAGCAGAGTTTTCAGCTAAACTGCCATTTCTGTCATAGTTTCAAGATTCACTCCGGCTCCATGTACTGGTAACTTACGATGTGAGAGACAGCTGTAACCAAGGTGAAGACAATCGATGCCTCTGAAGTGTGGTTCAAATAATTCCTTCAGGTCCCGACAATCAGTGAGTCCGTTCTCCGAGCTGAAGACACCCTGACAGTAACTCTGGGCGTGACCCTAAACATCGCTTCAGGAAGTGTGAATCACGACTTCACATCCTTTTTCTCTAGCATTCTTCTGTAAATAACAATCACTATTCATGGTCAAGAAATTAATTTTAAAAAGTMus musculas beta klotho protein (GenBank Accession No. NP_112457)(SEQ ID NO: 11)MKTGCAAGSPGNEWIFFSSDERNTRSRKTMSNRALQRSAVLSAFVLLAVTGSGDGKAWDKKQYVSPVNPSQLFLYDTFPKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTLYHWDLPLTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYWITIHNPYLVAWHGFGTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKGWLSITLGSHWIEPNRTDNMEDVINCQHSMSSVLGWFANPIHGDGDYPEFM KTGAMIPEFSEAEKEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNWIKLEYDDPQILISENGWFTDSYIKTEDTTAIYMMKNFLNQVLQAIKFDEIRVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSSAHYYKQIIQDNGFPLKESTPDMKGRFPCDFSWGVTESVLKPEFTVSSPQFTDPHLYVWNVTGNRLLYRVEGVRLKTRPSQCTDYVSIKKRVEMLAKMKVTHYQFALDWTSILPTGNLSKVNRQVLRYYRCVVSEGLKLGVFPMVTLYHPTHSHLGLPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINEPNRLSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLSLHCDWAEPANPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEYIASKNQRGLSSSVLPRFTAKESRLVKGTVDFYALNHFTTRFVIHKQLNTNRSVADRDVQFLQDITRLSSPSRLAVTPWGVRKLLAWIRRNYRDRDIYITANGIDDLALEDDQIRKYYLEKYVQEALKAYLIDKVKIKGYYAFKLTEEKSKPRFGFFTSDFRAKSSVQFYSKLISSSGLPAENRSPACGQPAEDTDCTICSFLVEKKPLIFFGCCFISTLAVLLSITVFHHQKRRKFQKARNLQNIPLKKGHSRVFS

Pharmaceutical Compositions

Another aspect of the disclosure pertains to pharmaceutical compositionsof the compounds of the disclosure. The pharmaceutical compositions ofthe disclosure typically comprise a compound of the disclosure and apharmaceutically acceptable carrier. As used herein “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible. Thetype of carrier can be selected based upon the intended route ofadministration. In various embodiments, the carrier is suitable forintravenous, intraperitoneal, subcutaneous, intramuscular, topical,transdermal or oral administration. Pharmaceutically acceptable carriersinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe pharmaceutical compositions of the disclosure is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be advantageous toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, monostearate salts and gelatin. Moreover, the compounds can beadministered in a time release formulation, for example in a compositionwhich includes a slow release polymer, or in a fat pad described herein.The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLG). Many methods for the preparation of such formulations aregenerally known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, certain methods of preparation are vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Depending on the route of administration, the compound may be coated ina material to protect it from the action of enzymes, acids and othernatural conditions which may inactivate the agent. For example, thecompound can be administered to a subject in an appropriate carrier ordiluent co-administered with enzyme inhibitors or in an appropriatecarrier such as liposomes. Pharmaceutically acceptable diluents includesaline and aqueous buffer solutions. Enzyme inhibitors includepancreatic trypsin inhibitor, diisopropylfluoro-phosphate (DEP) andtrasylol. Liposomes include water-in-oil-in-water emulsions as well asconventional liposomes (Strejan, et al., (1984) J. Neuroimmunol 7:27).Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations may contain a preservative toprevent the growth of microorganisms.

The active agent in the composition (i.e., long-acting FGF21) optionallyis formulated in the composition in a therapeutically effective amount.A “therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result to thereby influence the therapeutic course of aparticular disease state. A therapeutically effective amount of anactive agent may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of the agent toelicit a desired response in the individual. Dosage regimens may beadjusted to provide the optimum therapeutic response. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the agent are outweighed by the therapeutically beneficial effects.In another embodiment, the active agent is formulated in the compositionin a prophylactically effective amount. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

The amount of active compound in the composition may vary according tofactors such as the disease state, age, sex, and weight of theindividual. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Exemplary dosages of compounds (e.g., long-acting FGF21) of thedisclosure include e.g., about 0.0001% to 5%, about 0.0001% to 1%, about0.0001% to 0.1%, about 0.001% to 0.1%, about 0.005%-0.1%, about 0.01% to0.1%, about 0.01% to 0.05% and about 0.05% to 0.1%.

Exemplary or long-acting or stable forms of FGF21, as described herein,may have a half-life greater than that of the native or wild type FGF21.The half-life of long-acting FGF21 may be greater than the half-life ofnative FGF21 by at least 1 hour, at least 2 hours, at least 3 hours, at4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least8 hours, at least 9 hours, at least 10 hours, at least 11 hours, atleast 12 hours, at least 24 hours, at least 48 hours, at least 72 hours,at least 4 days, at least 5 days, at least 6 days, at least 7 days, atleast 2 weeks, at least 3 weeks, and at least a month, but at least someamount over that of the native FGF21.

Exemplary stabilized or long-acting forms of FGF21 (i.e., long-actingFGF21) as described herein, may have a half-life of at least 1.5×, atleast 2×, at least 3×, at least 4×, at least 5×, at least 8×, at least10×, at least 12×, at least 15×, at least 20×, at least 30×, at least40×, at least 50×, at least 60×, or at least 70× the half-life of anative FGF21 peptide, when assayed for stability under identicalconditions. Exemplary stabilized or long-acting forms of FGF21 (i.e.,long-acting FGF21) as described herein, may possesses a half-life of atleast 0.8 h, at least 1 h, at least 2 h, at least 3 h, at least 4 h, atleast 5 h, at least 7 h, at least 10 h, at least 15 h, at least 20 h, atleast 25 h, at least 28 h or at least 30 h in the circulation of amammal, optionally wherein the mammal is human.

The compound(s) of the disclosure can be administered in a manner thatprolongs the duration of the bioavailability of the compound(s),increases the duration of action of the compound(s) and the release timeframe of the compound by an amount selected from the group consisting ofat least 3 hours, at least 6 hours, at least 12 hours, at least 24hours, at least 48 hours, at least 72 hours, at least 4 days, at least 5days, at least 6 days, at least 7 days, at least 2 weeks, at least 3weeks, and at least a month, but at least some amount over that of thecompound(s) in the absence of the fat pad delivery system. Optionally,the duration of any or all of the preceding effects is extended by atleast 30 minutes, at least an hour, at least 2 hours, at least 3 hours,at least 6 hours, at least 12 hours, at least 24 hours, at least 48hours, at least 72 hours, at least 4 days, at least 5 days, at least 6days, at least 7 days, at least 2 weeks, at least 3 weeks or at least amonth.

A compound of the disclosure can be formulated into a pharmaceuticalcomposition wherein the compound is the only active agent therein.Alternatively, the pharmaceutical composition can contain additionalactive agents. For example, two or more compounds of the disclosure maybe used in combination. Moreover, a compound of the disclosure can becombined with one or more other agents that have modulatory effects oncancer.

Kits

The disclosure also includes kits that include a composition of thedisclosure, optionally also including a compound (e.g., a long-actingFGF21), and instructions for use.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents, and published patent applications cited throughout thisapplication, as well as the figures, are incorporated herein byreference.

EXAMPLES Example I: Materials and Methods Mouse Models ofRetinal/Choroidal Neovascularization

Oxygen-Induced Retinopathy:

mice were exposed to 75% oxygen from P7 to P12. The mice were thenreturned to room air until P17 (Connor et al., 2009; Smith et al.,1994). High oxygen led to vessel loss and the relative hypoxia inducedneovascularization which reached to maximum at P17 (Smith et al., 1994).Both genders of mouse pups were used with body weight ranging from 5.5to 7 grams (Connor et al., 2009). The mice were randomly assigned toeither native FGF21 (5 mg/kg, twice per day) or long-acting FGF21 (1mg/kg PF-05231023) or phosphate buffered saline (PBS). The mice wereintraperitoneally injected daily from P12 to P16. For the intra-vitrealinjection of PF-05231023 (0.5 μl 10 μg/μl stock per eye) was deliveredusing 10 μl syringe with 35G needle (World Precision Instruments, Inc.)at P12. The contralateral eye was injected with vehicle control. At P17,retinal neovascularization was quantified (Connor et al., 2009; Smith etal., 1994). Mice were sacrificed at P17 using a lethal intraperitonealinjection of ketamine/xylazine, and the eyes were enucleated, followedby fixation in 4% paraformaldehyde for 1 hour at room temperature. Theretinas were dissected and stained overnight at room temperature withfluorescent Griffonia Bandeiraea Simplicifolia Isolectin B4 (Alexa Fluor594, 121413, Molecular Probes, 10 μg/ml) in 1 mM CaCl2 in PBS. Theimages of whole mounted retina were taken at 5× magnification on a ZeissAxioObserver.Z1 microscope and merged to form one image with AxioVision4.6.3.0 software. Retinal neovascularization was quantified aspreviously reported (Connor et al., 2009). Percentages ofneovascularization were calculated by comparing the number of pixels inthe neovascular areas with the total number of pixels in the retina. nis the number of eyes quantified.

Vldlr^(−/−) Mice:

Vldlr^(−/−) mice develop pathological neovascularization (retinalangiomatous proliferation or RAP as well as later onset choroidalneovascularization). RAP extends from the deep retinal vascular layer ofthe outer plexiform layer (OPL) towards the retinal pigment epithelium(RPE). The mouse pups were treated with a long-acting FGF21 analogue(0.5 mg/kg PF-05231023) or PBS from P8 to P15. At P16, neovascularlesions were quantified (Joyal et al., 2016). The eyes were enucleatedand fixed in 4% paraformaldehyde for 1 hour at room temperature. Theretinas were stained overnight with fluorescent Griffonia BandeiraeaSimplicifolia Isolectin B4 (Alexa Fluor 594, 121413, Molecular Probes,10 μg/ml) in 1 mM CaCl2 in PBS then washed with PBS and whole mountedwith photoreceptors facing up. The images of whole mounted retina weretaken at 5× magnification on a Zeiss AxioObserver.Z1 microscope andmerged to form one image with AxioVision 4.6.3.0 software. Vascularlesions were analyzed using the SWIFT_MACTEL method, a plugin in Image J(Joyal et al., 2016).

Laser-Induced Choroidal Neovascularization:

Four laser burns were induced by a green Argon laser pulse, withduration of 70 ms and power of 240 mW in 6-8-week-old C57BL/6J mice.Both male and female mice were used (Lambert et al., 2013). Along-acting FGF21 analogue (10 mg/kg PF-05231023) or PBS wereintraperitoneally injected every other day one week before and after thelaser photocoagulation induction. The eyes were enucleated and fixed in4% paraformaldehyde for 1 hour at room temperature. The choroid werepenetrated with 1% Triton X-100 PBS for one hour at room temperature andstained overnight with fluorescent Griffonia Bandeiraea SimplicifoliaIsolectin B4 (Alexa Fluor 594, 121413, Molecular Probes, 10 μg/ml) in 1mM CaCl2 in PBS. The choroids were washed with PBS and whole mounted.The images of whole mounted choroid were taken at 10× or 20×magnification on a Zeiss AxioObserver.Z1 microscope. Lesion area wasquantified (Gong et al., 2015).

Real-Time PCR

RNA from retinas or choroid-retina complex was extracted andreverse-transcribed to cDNA. PCR was conducted for Fgfr1, Fgfr2, Fgfr3,Fgfr4, Klb, Apn, Vegfa, Dynamin2 and Tnfα. Cyclophilin A was used asinternal control. Freshly isolated retinas were lysed with QIAzol™ lysisreagent and incubated on ice for 15 minutes. 20% chloroform was addedand samples were incubated for 5 minutes at room temperature. RNA wasextracted according to the manufacturer's instructions using a PureLink®RNA Mini Kit (#12183018A, Ambion). RNA was then reverse transcribedusing iScript™ cDNA synthesis kit (#1708891, Bio-Rad). The sequences ofprimers were Fgfr1 ((F: 5′-ACT CTG CGC TGG TTG AAA AAT-3′ (SEQ ID NO:12), R: 5′-GGT GGC ATA GCG AAC CTT GTA-3′ (SEQ ID NO: 13)); Fgfr2 ((F:5′-GCT ATA AGG TAC GAA ACC AGC AC-3′ (SEQ ID NO: 14), R: 5′-GGT TGA TGGACC CGT ATT CAT TC-3′ (SEQ ID NO: 15)); Fgfr3 ((F: 5′-GCC TGC GTG CTAGTG TTC T-3′ (SEQ ID NO: 16), R: 5′-TAC CAT CCT TAG CCC AGA CCG-3′(SEQID NO: 17)); Fgfr4 ((F: 5′-TCC ATG ACC GTC GTA CAC AAT-3′ (SEQ ID NO:18), R: 5′-ATT TGA CAG TAT TCC CGG CAG-3′ (SEQ ID NO: 19)); β-klotho(Klb) ((F: 5′-TGT TCT GCT GCG AGC TGT TAC-3′ (SEQ ID NO: 20), R: 5′-CCGGAC TCA CGT ACT GTT TTT-3′ (SEQ ID NO: 21)); Adipoq: ((F: 5′-GAA GCC GCTTAT GTG TAT CGC-3′ (SEQ ID NO: 22), R: 5′-GAA TGG GTA CAT TGG GAA CAGT-3′ (SEQ ID NO: 23)); Vegfa ((F: 5′-GGA GAT CCT TCG AGG AGC ACT T-3′(SEQ ID NO: 24), R: 5′-GCG ATT TAG CAG CAG ATA TAA GAA-3′ (SEQ ID NO:25)); Tnfα ((F: 5′-AAG GAC CTG GTA CAT GAA CTG G-3′ (SEQ ID NO: 26), R:5′-GGT TCT GGG TGT CAA GTG TCG-3′ (SEQ ID NO: 27)); Dynamin 2 ((F:5′-TTT GGC GTT CGA GGC CAT T-3′ (SEQ ID NO: 28); R: 5′-CAG GTC CAC GCATTT CAG AC-3′ (SEQ ID NO: 29)) Quantitative analysis of gene expressionwas performed using an Applied Biosystems 7300 Sequence Detection Systemwith the SYBR Green Master mix kit, and gene expression was calculatedrelative to Cyclophilin A ((F: 5′-CAG ACG CCA CTG TCG CTT T-3′ (SEQ IDNO: 30); R: 5′-TGT CTT TGG AAC TTT GTC TGC AA-3′ (SEQ ID NO: 31)) usingthe ΔΔCt method.

ELISA for Mouse Serum FGF21

Neonatal mouse serum levels of FGF21 were measured with ELISA followingthe manufacturer's protocol (Eagle Biosciences, #F2131-K01). Briefly, 50μl mouse serum or standards were first added on the designatedmicrowell. 50 μl tracer antibody was then added and the plate wasincubated at room temperature for 2 hours. The wells were washed andincubated with 100 μl ELISA HRP substrate at room temperature in darkfor 20 minutes. Finally, 100 μl ELISA stop solution were added and thesignals were read at 450/650 nm.

Statistics

All data were used except for low quality images that were insufficientfor analysis. Data represent mean±SEM. 2-tailed unpaired t-test or ANOVAwith Bonferroni's multiple comparison test was used for comparison ofresults as specified (Prism v5.0; GraphPad Software, Inc., San Diego,Calif.). Statistically significant difference was set at P≤0.05. Forphenotypic data, a dot represents each retina or choroid; for qPCR data,each dot represents a number of replicates from 4-6 pooled retinas.

Example 2: Long-Acting FGF21 Suppressed Hypoxia-Induced RetinalNeovascularization

Early vessel growth cessation or vessel loss in retinopathy ofprematurity and diabetic retinopathy has been previously described tolead to hypoxia and nutrient deficits, which drives vessel overgrowth(Chen and Smith, 2007; Shin et al., 2014). To investigate the effects ofFGF21 on pathologic neovessel growth under hypoxia, FGF21 wasadministered to mice having oxygen-induced retinopathy (Smith et al.,1994). After five-day exposure to 75% oxygen, mouse pups with theirnursing dam were returned to room air. The oxygen exposure led tovaso-obliteration in the central retina, and the relatively avascularhypoxic retina induced neovascularization (Smith et al., 1994) extendingfrom the retina into the vitreous at the boundary between vascular andnon-vascularized areas (Connor et al., 2009). The mouse pups wereintraperitoneally injected with native FGF21 or a long-acting FGF21analog, PF-05231023 (Talukdar et al., 2016), or vehicle control for fivedays. Native FGF21, which exhibits a short half-life (0.4 hours; Huanget al., 2013), did not affect neovascularization (FIG. 1A) whereasadministration of PF-05231023, which possesses a biological half-life of28 hours (Huang et al., 2013) significantly decreased neovascularization(FIG. 1A). To confirm the role of FGF21 in retinal neovascularization,the impact of FGF21 deficiency on oxygen-induced retinopathy wasexamined by comparing wild-type (Fgf21^(+/+)) and knockout (Fgf21^(−/−))mice. Fgf21^(−/−) mice exhibited increased neovascularization (FIG. 1B).Noting the strong inhibition of pathologic neovessel formation observedwhen PF-05231023 was administered, the relatively smaller effects ofnative FGF21 and the effects of endogenous FGF21 deficiency observedupon retinal neovascularization, these latter effects were likely due tothe short half-life of native FGF21 (0.4 hours) (Huang et al., 2013) andlow endogenous FGF21 levels (about 658.3±66.4 pg/ml in normal neonatalmouse serum detected by ELISA), respectively, while the former effectsof long-acting FGF21 were also reflective of the short half-life ofnative FGF21. To determine if long-acting FGF21 directly inhibitedneovascularization, intra-vitreal injection of PF-05231023 wasperformed, and indeed was found to reduce retinal neovascularization, ascompared to the extent of retinal neovascularization observed in avehicle-injected contralateral eye (FIG. 5). FGF21 also promoted retinalrevascularization through APN, although APN might not be required for“basal” revascularization in OIR (FIG. 6A-6D). In some embodiments,improved revascularization has likely decreased the stimulus forproliferative neovascularization.

Without wishing to be bound by theory, in some embodiments, FGF21apparently exerted its effects through interaction with its receptor,FGFR1, and co-receptor β-klotho (KLB) (Ding et al., 2012; Foltz et al.,2012; Suzuki et al., 2008). As described herein, FGF21 receptor 1, 2, 3,4 and Klb mRNA were all expressed in the mouse retina (FIG. 2A). Fgfr1and fgfr3 were highly expressed in neovessels, and co-localized with APN(FIG. 7A-7B). PF-05231023 administration at P17 increased retinal Apn(FIG. 2B), an important mediator of FGF21 effects on metabolic function(Holland et al., 2013; Lin et al., 2013). APN receptor agonist AdipoRoninhibited endothelial cell function in vitro (FIG. 7C-7D). To determineif APN also mediated the protection conferred by long-acting FGF21against retinal neovascularization, the retinal vasculature in APNknockout (Apn^(−/−)) mice having oxygen-induced retinopathy wasexamined, in the presence or absence of PF-05231023 administration. APNdeficiency worsened retinal neovascularization (FIG. 2C), and lack ofAPN completely abolished the beneficial effects of PF-05231023 otherwiseexhibited in reducing neovascularization in hypoxic retinas (FIG. 2D).APN has been described as inhibiting retinal neovascularization bydecreasing the levels of tumor necrosis factor (TNF)α (Higuchi et al.,2009). Consistent with such observations, PF-05231023 suppressed Tnfαexpression in neovascular WT retina, but the suppression was abolishedwith APN deficiency (FIG. 2E). In some embodiments, long-acting FGF21inhibited pathological retinal neovessel growth by targeting APN andreducing TNFα (FIG. 2F), a key risk factor for oxygen-inducedretinopathy (Kociok et al., 2006).

Each year, over 15 million babies are born preterm and possessing anincompletely vascularized retina, which—if normal vascularization doesnot occur postnatally—sets the stage for the progression toproliferative retinopathy (Hellstrom et al., 2013). Retinopathy ofprematurity is a leading cause of blindness in children (Gilbert et al.,1997). As described herein, low serum APN levels positively correlatedwith proliferative retinopathy in premature infants (Fu et al., 2015).Increasing circulating APN levels were associated with less retinalneovascularization in mice (Fu et al., 2015; Higuchi et al., 2010). Inmouse oxygen-induced retinopathy, FGF21 administration increased retinalApn levels and reduced Tnfα levels, and also suppressed pathologicneovessel growth (FIG. 2).

Diabetic retinopathy currently afflicts approximately 93 million peopleworldwide, and of those, 28 million have vision-threateningproliferative retinopathy (Abcouwer and Gardner, 2014). The levels ofFGF21 in type 1 diabetes have been observed as lower than those inhealthy controls (Xiao et al., 2012; Zibar et al., 2014). Instreptozotocin-induced type 1 diabetic mice, a FGF21 analog reducedblood glucose levels with improved glucose uptake in brown adiposetissue (Kim et al., 2015). FGF21 prevented renal lipid accumulation, andattenuated renal dysfunction in type 1 diabetic mice (Zhang et al.,2013). In type 2 diabetes, serum FGF21 levels have been observed ashigher in patients with retinopathy versus no retinopathy (Esteghamatiet al., 2016; Lin et al., 2014). FGF21 treatment decreased body weightand improved the lipid profile (decreases triglycerides and increasesHDL cholesterol levels) in type 2 diabetes patients, non-human primatesand in obese rodents (Bernardo et al., 2015; Gaich et al., 2013; Schleinet al., 2016; Talukdar et al., 2016). It was hypothesized that FGF21could play a beneficial role in diabetes and diabetic complications,such as diabetic retinopathy. The mouse model of oxygen-inducedretinopathy has been commonly used to model hypoxia-inducedneovascularization in proliferative diabetic retinopathy (Lai and Lo,2013). As described elsewhere herein, long-acting FGF21 is alsocontemplated as helping to prevent proliferative diabetic retinopathy,optionally via a direct effect upon early diabetic retinal neurovascularloss and/or late neovascularization, optionally also related to thehyperglycemic aspect of diabetic retinopathy.

Example 3: Long-Acting FGF21 Administration Protected Against RetinalNeovascularization Induced by Energy-Deficiency in Vldlr^(−/−) Mice

In addition to lack of oxygen, an inadequate fuel supply can also driveretinal neovascularization (Joyal et al., 2016). Absence of the very lowdensity lipoprotein receptor (VLDLR) has previously been described asassociated with retinal angiomatous proliferation and choroidalneovascularization, similar to the neovascularization that has been seenwith macular telangiectasia and late proliferative age-related maculardegeneration (Engelbert and Yannuzzi, 2012; Lambert et al., 2016). InVldlr^(−/−) mice, abnormal blood vessels extended towards starvedphotoreceptors (Joyal et al., 2016) (FIG. 3A). To assess whetherlong-acting FGF21 protected against metabolism-induced pathologicneovessel growth, PF-05231023 was administered intraperitoneally at 0.5mg/kg daily from P8 to P15 to Vldlr^(−/−) mice. PF-05231023administration attenuated the neovascular lesions (FIG. 3A, 3B, 3C) andincreased retinal Apn, while decreasing Tnfα (FIG. 3D) in Vldlr^(−/−)mice. Thus, long-acting FGF21 administration protected against retinalneovascularization induced by energy-deficiency in Vldlr^(−/−) mice.

Example 4: Long-Acting FGF21 Inhibited Laser-Induced ChoroidalNeovascularization in Mice

Choroidal neovascularization, a condition in which neovessels extendfrom the choriocapillaris into the subretinal space, isvision-threatening in age-related macular degeneration. In alaser-induced choroidal neovascularization mouse model, laser burnsdisrupt Bruch's membrane to induce choroidal neovessel growth (Ryan,1979) (FIG. 4A). To test the effects of long-acting FGF21 in such amodel, 10 mg/kg PF-05231023 was administered every other day one weekbefore and after the laser injury in 6-8-week-old C57B/6J mice. As shownin FIG. 4B, PF-05231023 administration inhibited choroidal neovesselformation. In the mouse choroidal neovascularization model, PF-05231023also induced Apn and reduced Tnfα in the choroid-retina complex (FIG.4C).

Neovascularization more generally is a leading cause of vision loss inboth age-related macular degeneration and macular telangiectasia in thepopulation over age 50 years (Heeren et al., 2014; Yonekawa et al.,2015). Metabolic alterations in retinal pigment epithelial cells andphotoreceptors contribute to disease progression (Barron et al., 2001;Joyal et al., 2016). In Vldlr^(−/−) mice, which have an inadequate fuelsupply driving retinal neovascularization, long acting FGF21administration was observed to reduce neovascular lesion formation. Asdescribed herein, long-acting FGF21 administration likely attenuated thedevelopment of metabolically driven retinal neovascularization, modelingmacular telangiectasia and some aspects of neovascular age-relatedmacular degeneration. Furthermore, in laser-induced choroidalneovascularization modeling inflammatory aspects of neovascularage-related macular degeneration (Parmeggiani et al., 2012), FGF21 alsosuppressed pathological choroidal angiogenesis in adult mice.

In summary, there is an unmet need for effective treatment ofvision-threatening eye neovessel growth. As described herein,long-acting FGF21 inhibited retinal and choroidal neovascularizationmediated by APN in mice. APN suppresses TNFα transcription and mRNAstability in macrophages (Park et al., 2008; Wulster-Radcliffe et al.,2004). Inhibition of TNFα has been previously characterized as leadingto decreased retinal and choroidal neovascularization (Kociok et al.,2006; Shi et al., 2006), possibly through increased endothelial cellsprouting (Hangai et al., 2006; Sainson et al., 2008). Long-acting FGF21was observed herein to have promoted migration in HRMECs in vitro (FIG.7C). Different target systems are likely affected by long-acting FGF21with opposite angiogenic responses. As described herein, long-actingFGF21 inhibitory effects on retinal and choroidal neovascularizationwere identified as independent of VEGFA (FIG. 4D). It has previouslybeen reported in clinical data that little to no effect of long-actingFGF21 has been observed upon glycemic endpoints, which has temperedenthusiasm for potential further development of long-acting FGF21 in theclinic for metabolic diseases; however, the instant disclosure offers aphenotypic basis and some mechanistic insights into a new indicationwhere long-acting FGF21 is likely to provide an effective prophylacticand/or therapeutic against retinopathy, thereby providing anext-generation standard of care for patients with pathological vascularproliferation in retinopathy of prematurity, diabetic retinopathy,macular telangiectasia and age-related macular degeneration.

Example 5: FGF21 in Retinopathy of Prematurity

Hyperglycemia is a recently described risk factor for retinopathy ofprematurity (ROP) in premature infants. In streptozotocin (STZ)-inducedhyperglycemic ROP mouse model, retinal vascular development wasconfirmed as delayed. Specifically, FGF21 deficiency delayed normalretinal vascular network formation and worsened hyperglycemic ROP insuch mice. Native (nFGF21) and long-acting FGF21 (PF-05231023) treatmentwere both observed to have promoted retinal vessel growth inhyperglycemic ROP. The protection of FGF21 observed was also confirmedas dependent upon adiponectin (APN).

As seen in FIGS. 8A and 8B, FGF21 deficiency delayed normal retinalvascular development (FIG. 12A) and worsened hyperglycemic retinopathy(FIG. 8B) in neonatal mice. As seen in FIGS. 9A and 9B, FGF21 promotedretinal vascular development in hyperglycemic mouse neonates. Bothnative FGF21 (nFGF21; FIG. 9A) and long-acting FGF21 (PF-05231023, FIG.9B) administration improved retinal vascular growth. In someembodiments, FGF21 promoted and/or improved retinal vascular growth. Insome embodiments, long-acting FGF21 promoted and/or improved retinalvascular growth.

As seen in FIGS. 10A and 10B, FGF21 protection against hyperglycemicretinopathy was dependent on adiponectin (APN). As shown in FIG. 10A,APN deficiency completely abolished FGF21 effects on STZ-inducedhyperglycemic retinopathy. As demonstrated in FIG. 10B, FGF21 increasedAPN levels in serum. In some embodiments, FGF21 therefore preventedand/or reduced retinopathy of prematurity. In certain embodiments,long-acting FGF21 therefore prevented and/or reduced retinopathy ofprematurity.

Example 6: Long-Acting FGF21 in Retinitis Pigmentosa (RP)

As seen in FIG. 11, long-acting FGF21 increased the photoreceptor layerthickness (ONL) in photoreceptor-degenerating mice rd10. To determine ifFGF21 preserved photoreceptor function, rd10 mice are treated withlong-acting FGF21 analog PF-05231023. Retinal histology, retinalfunction with electroretinogram (ERG), and cone survival withimmunohistochemistry are used to assess preservation of photoreceptorfunction by long-acting FGF21. Retinal mitochondrial function andglycolysis with Seahorse Fx96 oxygen consumption rate (OCR) as well asan extracellular acidification rate (ECAR) analysis are also performedto assess preservation of photoreceptor function by long-acting FGF21.Furthermore, retinal key metabolic enzyme levels (Krebs cycle,glycolysis, fatty acid oxidation) are also performed with quantitativeproteomic analysis to assess preservation of photoreceptor function bylong-acting FGF21. Finally, mitochondrial function and glycolysis(Seahorse OCR, ECAR analysis) in cone-like photoreceptors (661W) invitro are also assessed in concert with PF-05231023 treatment.

To determine if FGF21 administration improves retinal vasculardevelopment in rd10, as retinal vessels reflect photoreceptor metabolicneeds, P30 deep vasculature in retinal whole mount in rd10 vs. controlmice with PF-05231023 treatment is quantified. Neovascular growth inlaser-captured microdissected retinal layers and vessels in WT and rd10mice, as well as PF-05231023 versus vehicle-treated rd10 mice are alsoquantified.

In some embodiments, these analyses and quantifications therebydemonstrate that long-acting FGF21 preserves photoreceptor function. Insome embodiments, these analyses and quantifications demonstrate thatlong-acting FGF21 improves retinal vascular development. Optionally,these analyses and quantifications demonstrate that long-acting FGF21prevents and/or reduces retinitis pigmentosa in a subject having or atrisk of developing RP.

Example 7: FGF21 Protects Against Early Diabetic Retinopathy (DR)

Retinal neuronal abnormalities occur before vascular changes in diabeticretinopathy. Accumulating experimental evidence suggests that neuronscontrol vascular pathology in diabetic and other neovascular retinaldisease. Whether fibroblast growth factor 21 (FGF21) prevented retinalneuronal dysfunction in insulin-deficient diabetic mice wasinvestigated.

Materials and Methods

Animals: All animal studies adhered to the Association for Research inVision and Ophthalmology Statement for the Use of Animals in Ophthalmicand Vision Research and were approved by the Institutional Animal Careand Use Committee at Boston Children's Hospital.

Mouse Models of Type 1 Diabetes—Akita Mice:

Ins2^(Akita) male mice have a spontaneous mutation in the insulin 2 genewhich leads to incorrect folding of insulin protein. Heterozygous Akitamice develop diabetes within one month and retinal complications around6-months of age (Lai A K, Lo A C. Journal of Diabetes Research2013:106594). 7-to-8-month-old Akita mice were compared with littermatewild-type (WT) mice. Akita mice with retinal functional abnormalitieswere screened with electroretinography (ERG) and those with ERG changeswere intraperitoneally injected with 10 mg/kg long-acting FGF21 analog,PF-05231023 (Pfizer) or vehicle control twice a week for four weeks.Retinal function was again examined with ERG after 4 weeks of treatmentand body weight, blood glucose were recorded. Serum triglycerides levelswere measured using the Wako L-Type TG M test.

Streptozotocin (STZ)-Induced Diabetic Mice:

6-to-8-week-old male WT and adiponectin-deficient (Apn^(−/−)) mice werestarved six hours prior to an intraperitoneal injection of 55 mg/kg/daySTZ on day 1 and 2 followed by an injection of 60 mg/kg/day STZ from day3 to 5. Diabetes was induced within one week of injection (Lai A K etal. 2013). 7-to-8-month-old diabetic WT and Apn^(−/−) mice were screenedwith ERG. Mice with retinal functional abnormalities wereintraperitoneally injected with 10 mg/kg PF-05231023 or control twice aweek for four weeks. Retinal function was then re-examined with ERG.

Electroretinography (ERG):

ERG was used to assess the function of retinal neurons. Flash ERGs wereobtained using an Espion e² (Diagnosys LLC, Lowell, Mass.) indark-adapted, mydriatic (Cyclomydril; Alcon, Fort Worth, Tex.),anesthetized (ketamine/xylazine) subjects. Stimuli were “green” lightemitting diode flashes of doubling intensity from ˜0.0064 to ˜2.05cd□s/m² and then “white” xenon-arc flashes from ˜8.2 to ˜1,050 cd□s/m²presented in an integrating sphere (Colordome, Diagnosys LLC). As shown(FIG. 14A), the saturating amplitude (Rm_(P3)) and sensitivity (S) ofthe rod photoreceptors was estimated from the a-waves elicited by thewhite flashes (Lamb T D, Pugh E N, Jr., J Physiol 1992; 449:719-758).The saturating amplitude (Rm_(P2)) and sensitivity (1/K_(P2)) ofsecond-order neurons, principally bipolar cells (Wurziger K, et al.,Vision Res 2001; 41:1091-1101), was measured by subtracting this modelfrom the intact ERG waveform (Robson J G, et al., Vis Neurosci 1995;12:837-850) and fitting the Naka-Rushton equation (Fulton A B, Rushton WA. Vision Research 1978; 18:793-800) to the response vs. intensityrelationship of the resulting waveform, “P2.” The oscillatory potentials(OPs), which characterize activity in inner retinal cells distinct fromthe generators of the a- and b-waves (Dong C J, et al., Vis Neurosci2004; 21:533-543), were filtered from P2 (Lei B, et al., InvestOphthalmol Vis Sci 2006; 47:2732-2738) and assessed in the frequencydomain to determine their energy (Akula J D, et al., Invest OphthalmolVis Sci 2007; 48:5788-5797); the saturating energy (Em) and sensitivity(1/i_(1/2)) of the OPs were then assessed similarly to those of P2.Finally, retinal sensitivity at threshold (Sm), was calculated byscaling the amplitude of each P2 by the intensity used to elicit it andfitting a generalized logistic growth curve, with the exponent set tonegative unity, to the resulting sensitivities, and determining thelimit of this function as intensity approached zero (Akula J D, et al.,Mol Vis 2008; 14:2499-2508).

Optical Coherence Tomography (OCT):

Mice were anesthetized (ketamine/xylazine), and their pupils dilated(Cyclomydril; Alcon, Fort Worth, Tex.). Spectral domain OCT withguidance of bright-field live fundus image was performed with theimage-guided OCT system (Micron IV, Phoenix Research Laboratories).Photoreceptor inner and outer segment thickness was measured usingInsight (provided by Micron IV). The thickness of photoreceptor segmentswas plotted at six distances (50, 100, 150, 200, 250 and 300 μm) fromthe optic nerve head both on the nasal and on the temporal side.

Immunohistochemistry:

For IHC on retinal cross-sections, eyes were fixed in 4% PFA, frozen inoptimal cutting temperature compound (OCT, Tissue-Tek) and then were cutinto 10-μm sections and rinsed with PBS. The sections with optic nervewere treated with ice-cold methanol for 15 minutes and then 0.1% tritonPBS for 45 minutes at room temperature. The sections were blocked with3% bovine serum albumin (BSA) for 1 hour at room temperature and stainedwith primary antibody against cone arrestin (1:500, AB15282, Millipore),rhodopsin (1:500, MABN15, Millipore) overnight at 4° C. The sectionswere stained with corresponding secondary antibody, covered in mountingmedium with 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI,H-1200, Vector laboratories), and visualized with a Leica SP2 confocalmicroscope or a Zeiss AxioObserver.Z1 microscope at 200× magnification.

Laser-Captured Microdissection:

Fresh mouse eyes were embedded in OCT compound and sectioned at 12 μm ina cryostat, mounted on RNase-free polyethylene naphthalate glass slides(Ser. No. 11/505,189, Leica). Frozen sections were fixed in 70% ethanolfor 15 seconds, followed by 30 seconds in 80% ethanol and 30 seconds inabsolute ethanol, and then washed with DEPC-treated water for 15seconds. Laser-dissection of retinal neuronal layers was performedimmediately thereafter with the Leica LMD 6000 system (LeicaMicrosystems) and samples were collected directly into lysis buffer fromthe RNeasy Micro kit (Qiagen, Chatsworth, Calif.).

Photoreceptors (661W) Cell Culture:

Photoreceptor 661W cells were cultured at 37° Celsius, 5% CO₂ in ahumidified atmosphere in Dulbecco's modified Eagle's medium (DMEM,#1196502, Gibco) supplemented with 10% fetal bovine serum (#S12450,Atlanta Biologicals) and 1% antibiotic/antimycotic solution. An equalnumber of cells per well was plated on a 6-well dish. Oxidative stresswas induced with 0.5 mM paraquat (PQ, Sigma, #856177) for 1 hour at 37°Celsius. The culture media were changed and the cells were treated withvehicle (PBS) or 500 ng/ml PF-05231023 for 24 hours. Cells werecollected for protein and RNA.

Real-Time PCR:

Retinas or 661W cells were lysed with QIAzol lysis reagent and incubatedon ice for 15 minutes. 20% chloroform was added and incubated for 5minutes at room temperature. RNA was extracted according to themanufacturer's instructions using a PureLink® RNA Mini Kit (#12183018A,Ambion). RNA was then reverse transcribed using iScript™ cDNA synthesiskit (#1708891, Bio-Rad). qPCR were performed for Arrestin4: 5′-GAG CAAGGG CTG CTA CTC AAG-3′ (SEQ ID NO: 32), 5′-AAC CGC AGG TTC AAG TATTCC-3′ (SEQ ID NO: 33); Rhodopsin: 5′-TCA TGG TCT TCG GAG GAT TCA C-3′(SEQ ID NO: 34), 5′-TCA CCT CCA AGT GTG GCA AAG-3′ (SEQ ID NO: 35);IL-1β: 5′-TTC AGG CAG GCA GTA TCA CTC-3′ (SEQ ID NO: 36), 5′-GAA GGT CCACGG GAA AGA CAC-3′ (SEQ ID NO: 37); IL-6: 5′-AAG AGC CGG AAA TCC ACGAAA-3′ (SEQ ID NO: 38), 5′-GTC TCA AAA GGG TCA GGG TAC T-3′ (SEQ ID NO:39); Vegfa F: 5′-GGA GAT CCT TCG AGG AGC ACT T-3′ (SEQ ID NO: 24), R:5′-GCG ATT TAG CAG CAG ATA TAA GAA-3′ (SEQ ID NO: 25); Tnfα 5′-AAG GACCTG GTA CAT GAA CTG G-3′ (SEQ ID NO: 26), 5′-GGT TCT GGG TGT CAA GTGTCG-3′ (SEQ ID NO: 27); IL-10: 5′-CTT ACT GAC TGG CAT GAG GAT CA-3′ (SEQID NO: 40), 5′-GCA GCT CTA GG AGC ATG TGG-3′ (SEQ ID NO: 41); Apn:5′-GAAGCCGCTTATGTGTATCGC-3′ (SEQ ID NO: 42),5′-GAATGGGTACATTGGGAACAGT-3′ (SEQ ID NO: 43); Nrf2: 5′-TAG ATG ACC ATGAGT CGC TTG C-3′ (SEQ ID NO: 44), 5′-GCC AAA CTT GCT CCA TGT CC-3′ (SEQID NO: 45); Nfκb: F: 5′-GGA GAG TCT GAC TCT CCC TGA GAA-3′ (SEQ ID NO:46), R: 5′-CGA TGG GTT CCG TCT TGG T-3′ (SEQ ID NO: 47). Quantitativeanalysis of gene expression was generated using an Applied Biosystems7300 Sequence Detection System with the SYBR Green Master mix kit andgene expression was calculated relative to Cyclophilin A ((5′-CAG ACGCCA CTG TCG CTT T-3′ (SEQ ID NO: 30); 5′-TGT CTT TGG AAC TTT GTC TGCAA-3′(SEQ ID NO: 31)) (retinas) or β-actin ((5′-CGG TTC CGA TGC CCT GAGGCT CTT-3′ (SEQ ID NO: 48), 5′-CGT CAC ACT TCA TGA TGG AAT TGA-3′ (SEQID NO: 49)) (photoreceptor 661W cells) using the ΔΔCt method. Eachsample was repeated in triplicate.

Western Blot:

5 μl protein lysate from photoreceptor 661W cells were used to detectthe levels of NRF2 (1:500, R&D, MAB3925), phospho-NFκB (1:200, CellSignaling, #3037S), NFκB (1:1000, Cell Signaling, #3034), p-AKT (1:500,Cell Signaling, #9271), AKT (1:1000, Cell Signaling, #4691) in 5% bovineserum albumin (BSA) overnight at 4° Celsius degree. Signals weredetected using 1:5000 corresponding horseradish peroxidase-conjugatedsecondary antibodies and enhanced chemiluminescence (ECL, Pierce), thenthe digital images were visualized with Bio-Rad ChemiDoc Touch ImagingSystem. β-ACTIN (1:5000, Sigma; A1978) was used as internal control.

Statistical Analysis:

All ERG data were presented as the log change from control (Δ LogNormal); by expressing the data in log values, changes in observationsof fixed proportion become linear, consistent with a constant fractionfor physiologically meaningful changes in parameter values (Akula J D,et al., Mol Vis 2008; 14:2499-2508). Δ Log Normal ERG data were plottedas mean±SEM and evaluated for significant effects using mixed-effectslinear models (MLMs) (Fitzmaurice G M, Laird N M, Ware J H: Appliedlongitudinal analysis. Hoboken, N.J., Wiley, 2011). In each analysis,two MLMs were employed. The first MLM, carried out on the saturatinga-wave, P2 and OP parameters, had factors group (Akita vs. WT; or STZvs. Control), treatment (before vs. after PF-05231023), parameter(Amplitude vs. Sensitivity), and retinal depth (photoreceptor vs.bipolar vs. inner retina). The second MLM, carried out on Sm, hadfactors group, and treatment. Data from both eyes was included in allanalyses. Differences in ERG parameters were detected by ANOVA followedby Tukey's test. Two-tailed unpaired t-test, ANOVA with Bonferroni'smultiple comparison test was used for comparison of results as specified(Prism v5.0; GraphPad Software, Inc., San Diego, Calif.). The thresholdfor statistical significance (a) was set at 0.05.

Results

PF-05231023 Administration Restored Retinal Function in Akita Mice:

Circulating FGF21 levels (ELISA) were reduced in >7 month old Akita miceversus littermate wild-type (WT) control mice (FIG. 13A). Retinalexpression of Fgf21 (qPCR) was not changed significantly (FIG. 13B). Thephysiologic and pharmacologic actions of FGF21 are dependent on thereceptor FGFR1 and co-receptor β-klotho (Foltz I N, et al., ScienceTranslational Medicine 2012; 4:162ra153; Ding X, et al., Cell Metabolism2012; 16:387-393). Gene expression of FGF21 receptor Fgfr1 wascomparable and β-klotho was mildly increased in Akita versus WT mouseretinas (FIGS. 13C-13D) (Foltz I N, et al., Science TranslationalMedicine 2012; 4:162ra153; Ding X, et al., Cell Metabolism 2012;16:387-393). Fgfr1 was expressed in retinal neurons isolated fromretinal cross sections with laser-captured microdissection (FIG. 20).

To examine if FGF21 protects against retinal dysfunction in DR,PF-05231023 (10 mg/kg intraperitoneally was first administered, twice aweek for a month) or vehicle control to those Akita mice which had ERGdeficits at 7-8-month of age (50% of Akita mice (6/12) had ERG deficitsversus their age-matched WT control mice). In Akita mice with ERGdeficits, PF-05231023 administration did not change the body weight andserum triglyceride levels versus controls but mildly reduced bloodglucose levels (FIGS. 14A-14C). Prior to PF-05231023 treatment, ERGresponses were examined in Akita mice. Although the Akita mouseresponses were slightly attenuated overall compared to WT controls(FIGS. 14A-14C), retinal sensitivity (Sm), as determined by mixed-linearmodeling, was significantly attenuated (F=15.9; df=1,27.0; p=0.00046)relative to WT. PF-05231023 showed protective effects on Akita retinas(FIGS. 14B-14C). Notably, retinal sensitivity (Sm) in Akita mice wasimproved following treatment (FIG. 14D) (F=27.9, df=1,29.8, p=1.1×10⁻⁵)to levels that were supranormal, a result of better a-wave (5) andb-wave (1/K_(P2)) sensitivities (FIG. 14E). There was no decline inbaseline retinal function (ERG signals) in any individual mouse between7 and 8 months of age (F=0.166, df=1,2, p=0.723, FIG. 22). In Akitamice, the change in post-receptor sensitivity (log 1/K_(P2)) waspositively correlated with the sum of changes in photoreceptorsensitivity and saturated amplitudes (log S+log Rm_(P3), a-wave) (FIG.15A), providing evidence that the changes in post-receptor cells werereflecting the deficits in photoreceptor function (Hansen R M, et al.,Progress in Retinal and Eye Research 2017; 56:32-57; Hood D C, et al.,Visual Neuroscience 1992; 8:107-126).

PF-05231023 administration restored photoreceptor morphology in Akitamice: In addition to neuronal function, cone and rod photoreceptorstructure was examined to determine if it was influenced by PF-05231023administration. PF-05231023 administration increased cone-specificarrestin4 expression (Craft C M, et al., The Journal of BiologicalChemistry 1994; 269:4613-4619) and did not change rhodopsin expressionat the mRNA levels in Akita mice (FIG. 15B). Cone photoreceptor outerand inner segments were oriented in parallel in WT mice but disorganizedin Akita mice. PF-05231023 administration normalized the photoreceptorsegment arrangement (FIG. 15C). Rhodopsin staining was comparablebetween WT and PF-05231023-treated Akita mouse retinas, while thereappeared to be a reduction in the thickness of rod photoreceptorsegments in Akita mice (FIG. 15D). With OCT measurements, there was asignificant reduction in the total thickness of photoreceptor inner andouter segment in Akita (blue line) versus WT (black line) mice, but theinner and outer segment thickness was restored with PF-05231023administration (orange line), particularly the photoreceptor outersegments (FIG. 15E). These observations provide evidence thatPF-05231023 protection against DR is through the restoration ofphotoreceptor function and structure. The possibility of thecontribution from other retinal cells was not excluded as Fgfr1 is alsoexpressed in INL and RGC (FIG. 20).

PF-05231023 Decreased Retinal Inflammation in Diabetic Mice:

Retinal inflammation induces retinal neurovascular abnormalities indiabetes (Du Y, et al., Proc. Nat'l. Acad. Sci. USA 2013;110:16586-16591; Liu H, et al., Inv. Ophthal. & Vis. Sci. 2016;57:4272-4281; Tonade D. et al., Invest. Ophthal. & Vis. Sci. 2016;57:4264-4271; Joussen A M, et al., FASEB J. 2004; 18:1450-1452).Significantly increased retinal IL-1β and decreased Vegfa mRNAexpression was observed in Akita versus WT mice (FIG. 16A). PF-05231023administration reduced retinal IL-1β mRNA expression in Akita mice (FIG.16B). IL-1β inhibits energy production in retinal neurons and inducesretinal microvascular changes in rats. PF-05231023 administration didnot change the expression levels of Vegfa, Tnfα, IL-6, IL-10 and Apn inAkita mouse retinas (FIG. 16B).

PF-05231023 Inhibited Oxidative-Stress-Induced Inflammation inPhotoreceptors:

Hyperglycemia induces oxidative stress, a crucial contributor todiabetic retinopathy (Madsen-Bouterse S A, Kowluru R A. Reviews inEndocrine & Metabolic Disorders 2008; 9:315-327). Photoreceptors are themost metabolically active cell in the body and very susceptible tometabolic derangement and resulting oxidative stress (Kern T S,Berkowitz B A. J. Diabetes Invest. 2015; 6:371-380). Modulatingphotoreceptor oxidative stress protects against retinalneurodegeneration (Xiong W, et al., J. Clin. Invest. 2015;125:1433-1445). In PF-05231023-versus vehicle-treated Akita mouseretinas, there was a significant increase in total AKT levels althoughthere was no significant change in the ratio of p-AKT/AKT (FIG. 17A),evidencing that the absolute level of p-AKT was higher inPF-05231023-treated mouse retinas. Activation of the AKT pathwayregulates NRF2 activity in retinal pigment epithelium in vitro (Wang L,et al., Invest. Ophthal. & Vis. Sci. 2008; 49:1671-1678). There was alarge variation of NRF2 protein levels in Akita versus WT mice.PF-05231023 administration decreased the variability of retinal NRF2levels in Akita (FIG. 17A). Taken together, the results provide evidencethat PF-05231023 modulates retinal NRF2 levels by activating the AKTpathway. To test if PF-05231023 protects photoreceptors againstoxidative stress, oxidative stress was induced with the use of paraquat(PQ), a nonselective herbicide to induce the production of reactiveoxygen species in mitochondria (McCarthy S, et al., Toxicol. AppliedPharma. 2004; 201:21-31). In 661W cells (the only photoreceptor cellline available currently) in vitro, oxidative stress induced with PQincreased IL-1β expression; PF-05231023 treatment prevented IL-1βinduction (FIG. 17B). Both the activation of antioxidant transcriptionalfactor NRF2 and the phosphorylation of NFκB modulate IL-1βtranscription, which can be modulated by FGF21 (Yu Y, et al.,International Immunopharmacology 2016; 38:144-152; Kobayashi E H, etal., Nature Communications 2016; 7:11624; Cogswell J P, et al., JImmunol 1994; 153:712-723). PF-05231023 treatment increased geneexpression of Nrf2 but not Nfκb in 661W (FIG. 17C). In PQ (to induceoxidative stress)-treated 661W cells, the induction of NRF2 expressionby PF-05231023 was dose-dependently inhibited by perifosine, an AKTinhibitor (Zitzmann K, et al., Endocrine-Related Cancer 2012;19:423-434) (FIG. 17D). PF-05231023 treatment also increased NRF2production at the protein level but did not change NFκB phosphorylationin photoreceptors with PQ-induced oxidative stress (FIG. 17E), providingevidence that FGF21 inhibition of IL-1β was through activation of theNRF2 pathway.

PF-05231023 Administration Protected STZ-Induced Diabetic Mice AgainstDR, Partly Independent of Adiponectin (APN):

APN is a key mediator of FGF21 modulation of glucose and lipidmetabolism in mice (Lin Z, et al. Cell Metabolism 2013; 17:779-78947;Holland W L, et al., Cell Metabolism 2013; 17:790-797). Changes in theAPN pathway may contribute to the development of neovascular eyediseases (Fu Z, et al., Biochimica et Biophysica Acta 2016;1862:1392-1400). To test if APN mediated the protective effects ofPF-05231023, diabetes was induced with injection of STZ in 6-8-week-oldWT and in APN-deficient (Apn^(−/−)) mice. Retinal function was thenexamined by ERG at 7-8-months of age. Again, PF-05231023 administrationdid not change body weight, blood glucose levels or serum triglyceridelevels (FIGS. 23A-23C). Furthermore, neither the amplitude norsensitivity of the a-wave, b-wave, or the OPs differed significantlybetween STZ-treated mice (FIGS. 18A-18C) but, retinal sensitivity atthreshold, Sm, was significantly attenuated following STZ-treatment(F=12.2, df=1,6.0, p=0.013, FIG. 18D). PF-05231023, administered asabove, again improved Sm (F=45.2, df=1,5.9, p=0.001) in the STZ treatedmice to levels that were supranormal (FIGS. 18D-18E). The protectiveeffects of PF-05231023 on retinal sensitivity (Sm) in STZ-induced WTdiabetic mice were again found in Apn diabetic mice (F=23.8, df=1,2,p=0.040, FIGS. 18F-18H), providing evidence that the rescue was partlyindependent of APN. In the STZ-induced diabetic mice, PF-05231023decreased IL-1β expression in diabetic WT and Apn^(−/−) retinas (FIG.18I), suggesting that PF-05231023-induced reduction in IL-1β wasindependent of APN, in line with the phenotypic observation above (FIGS.18F-18H).

DISCUSSION

Dysfunction in photoreceptors and post receptor neurons are among theearly retinal changes seen in diabetic patients, antecedingophthalmoscopic signs of retinopathy (Pescosolido N. et al., J. DiabetesResearch 2015; 2015: 319692). In diabetes, hyperglycemia inducesoxidative stress, a crucial contributor leading to diabetic retinopathy(Madsen-Bouterse S A, Kowluru R A.: Oxidative stress and diabeticretinopathy: pathophysiological mechanisms and treatment perspectives.Reviews in Endocrine & Metabolic Disorders 2008; 9:315-327). Activationof the anti-oxidant protein NRF2 protects against retinal neuronaldegeneration (Xiong W., et al. The Journal of Clin. Invest. 2015;125:1433-1445), particularly in photoreceptors, as photoreceptors arethe most metabolically active cells in the body (Wong-Riley M T. Eye andBrain 2010; 2:99-116; Okawa H, et al., Current Biology: CB 2008;18:1917-1921) and more vulnerable to oxidative stress damage. It wasdemonstrated herein that, in insulin-deficient diabetic mice,administration of the long-acting FGF21 analog PF-05231023 reverseddiabetes-induced retinal neuronal deficits with improved photoreceptorfunction and morphology, and decreased photoreceptor-derivedinflammation (FIG. 19). PF-05231023 administration regulated retinalNRF2 levels through activation of the AKT pathway, and suppression ofpro-inflammatory IL-1β expression. IL-1β causes neurovascular damages inthe retina (Abcouwer S F, et al., Invest. Ophthal. & Vis. Sci. 2008;49:5581-5592; Kowluru R A, Odenbach S. Invest. Ophthal. & Vis. Sci.2004; 45:4161-4166; Liu Y. et al., PloS one 2012; 7:e36949). Therefore,it was proposed that FGF21 (PF-05231023) regulates retinal NRF2 levelsto reduce IL-1β production and photoreceptor dysfunction in DR.

Photoreceptor high energy consumption makes it susceptible toneurovascular disease. Blood vessels supply nutrients and oxygen toneurons, and evacuate waste. Disturbances in neuronal activity triggervascular remodeling (Fulton A B, et al., Documenta OphthalmologicaAdvances in Ophthalmology 2009; 118:55-61). In diabetic animal models,photoreceptor responses to hyperglycemia induce retinal blood vesselloss (Du Y. et al., Proc. Nat'l. Acad. Sci. USA 2013; 110:16586-165916;Liu H., et al., Invest. Ophthal. &Vis. Sci. 2016; 57:4272-4281; TonadeD. et al., Invest. Ophthal. & Vis. Sci. 2016; 57:4264-4271). Inaddition, low rod sensitivity is associated with abnormal retinalvasculature. Rod photoreceptor demands contribute to the vascularrecovery in hypoxia-induced retinal neovascularization (Akula J D etal., Invest. Ophthal. & Vis. Sci. 2007; 48:4351-4359). Photoreceptormetabolic dysfunction dictates pathological retinal angiogenesis (JoyalJ S et al., Nature Medicine 2016; 22:439-445). Therefore, maintainingphotoreceptor function may prevent vascular abnormalities in DR. Ininsulin-deficient Akita mice, reduced sensitivity was found in thepost-receptor retina, in line with clinical observations (PescosolidoN., et al., J. Diabetes Res. 2015; 2015:319692). Meanwhile, the changesin post-receptor cells were actually reflecting the deficits inphotoreceptor function (Hansen R M, et al., Prog. Retinal and Eye Res.2017; 56:32-57; Hood D C, et al. Vis. Neurosci. 1992; 8:107-126).Administration of a FGF21 analogue, PF-05231023, reversed thediabetes-induced morphological changes in photoreceptors and restoredretinal sensitivity; it also reduced disorganization of thephotoreceptor segments. This provides evidence that PF-05231023″s effecton retinal function ERG may be due to improved photoreceptor structureand function.

There is a strong correlation between hyperglycemia and the developmentof DR. Hyperglycemia leads to many cellular metabolic alterations thatcould serve as therapeutic targets. However, while pharmacologicinterventions that disrupt putative biochemical signaling pathwaysbetween hyperglycemia and DR, an effective and safe drug is not yetavailable. Type 1 diabetic patients have low circulating FGF21 levels(Zibar K, et al., Endocrine 2014; Xiao Y, et al., J. Clin. Endocrinol.Metabol. 2012; 97:E54-58) and FGF21 administration reduces hyperglycemiaand lessens renal dysfunction in type 1 diabetic mice (Jiang X, et al.,Toxicol. Letters 2013; 219:65-76). FGF21 also improves the lipid profile(decreased triglycerides) of obese monkeys and type 2 diabetic patients(Talukdar S, et al., Cell Metabolism 2016; 23:427-440), indicating thatFGF21 may have positive effects on diabetes and diabetic complications.

No significant impact of PF-05231023 administration on serumtriglyceride levels in either Akita or STZ-induced diabetic mice.Although PF-05231023 slightly reduced hyperglycemia in Akita mice, thisfinding was not replicated in STZ-induced mice. The protective effectsof PF-05231023 on retinal neurons is, therefore, likely to beindependent of circulating glucose and lipid modulation. It was alsodiscovered that PF-05231023 protection against retinal neuronal deficitswas preserved with APN deficiency. In the oxygen-induced retinopathymouse model of late vaso-proliferative retinopathy, FGF21 inhibitspathologic neovessel growth mediated by APN (Fu Z, et al., Cell Reports2017; 18:1606-1613). The current findings indicate that FGF21 regulatesretinal neuron and neovessel growth through other mechanisms.

Oxidative stress resulting from highly metabolic photoreceptors inducesinflammation, which induces DR (Du Y, et al., Proc. Nat'l. Acad. Sci.USA 2013; 110:16586-16591; Liu H, et al., Inv. Ophthal. & Vis. Sci.2016; 57:4272-4281; Tonade D. et al., Invest. Ophthal. & Vis. Sci. 2016;57:4264-4271; Joussen A M et al., FASEB J. 2004; 18:1450-1452; Kern T S,Berkowitz B A: J. Diabetes Invest. 2015; 6:371-380). Modulatingoxidative stress prevents the progression of DR (Williams M, et al.,Current Diabetes Reports 2013; 13:481-487). PF-05231023 administrationattenuated the diabetes-induced IL-1β expression in Akita mice. FGF21reduces oxidative stress and inhibits the NFκB pathway in mice (Yu Y, etal., International Immunopharmacology 2016; 38:144-152). Inphotoreceptors in vitro with paraquat-induced oxidative stress, it wasobserved that PF-05231023 treatment decreased IL-1β expression throughthe activation of the NRF2 pathway, which is known for its antioxidantcapability (Xiong W, et al., J. Clin. Invest. 2015; 125:1433-1445) andregulation of IL-1β transcription (Yu Y, et al., 2016; Kobayashi E H, etal., Nature Communications 2016; 7:1162443; 44). Additionally,PF-05231023-induced effect on IL-1β was independent of APN in diabeticretinas, in line with the neuronal observation. APN inhibits retinalneovessel growth via TNFα (Higuchi A, et al., Circulation Research 2009;104:1058-1065) and FGF21 also reduces TNFα in neovascular mouse retinas(Fu Z, et al., 2017). However, in Akita mice, there was no significantchange in retinal Tnfα expression between PF-05231023- andvehicle-treated groups. Taken together, it was concluded that indiabetic retinas, PF-05231023 protected neuronal activity through theNRF2-IL-1β pathway, which was at least to some degree independent of theAPN-TNFα pathway that the inventors showed to be involved in retinalneovascularization in OIR (Fu Z, et al., Am. J. Clin. Nutrition 2015;101:879-888). IL-10 causes a decline in mitochondrial membrane potentialand ATP production in retinal neurons (Abcouwer S F, et al., Invest.Ophthal. & Vis. Sci. 2008; 49:5581-5592). Reduction of retinal IL-10 mayprevent the induction of early vessel loss in DR as IL-1β inducesretinal microvascular abnormalities in rats (Kowluru R A, Odenbach S.Invest. Ophthal. & Vis. Sci. 2004; 45:4161-4166; Liu Y, et al., PloS one2012; 7:e36949).

In this study, PF-05231023 was administrated intraperitoneally andcirculating FGF21 levels were measured. PF-05231023 administration didnot alter retinal Fgf21, Fgfr1 and Klb expression in Akita mice (FIGS.24A-24C). These data implicate circulating/peripheral FGF21 as a primarydriver of retinal protection rather than autocrine/paracrine effects ofFGF21 in the retina. Although FGF21 is expressed in liver, white adiposetissue and brown adipose tissue, it is a hepatokine and liver is theprimary source of circulating FGF21 under fasting and refeedingconditions (Markan K R, et al., Diabetes 2014; 63:4057-4063). In humans,liver is also the primary source of circulating FGF21 in a patternconsistent with a hormonal response (Yang C, et al., BMCGastroenterology 2013; 13:67). While liver-derived FGF21 is critical forthe adaptive response to fasting or starvation in rodents, in humans,FGF21 plays an important role in fructose metabolism (Dushay J R, et al.Molecular Metabolism 2015; 4:51-57). Circulating FGF21 has been shown tocross the blood brain barrier in humans in a non-saturable,unidirectional manner (Hsuchou H, et al., Peptides 2007; 28:2382-2386).FGF21 regulates metabolism and circadian behavior, sweet and alcoholpreferences by directly acting on the nervous system (Bookout A L, etal. Nature Medicine 2013; 19:1147-1152; Talukdar S, et al. CellMetabolism 2016; 23:344-349). The blood retinal barrier (BRB), which isessential for normal visual function (Cunha-Vaz J: The Blood-RetinalBarrier in the Management of Retinal Disease: EURETINA Award Lecture.Ophthalmologica Journal international d'ophtalmologie Internationaljournal of ophthalmology Zeitschrift fur Augenheilkunde 2017; 237:1-10),is broken down in DR (Klaassen I, et al. Progress in Retinal and EyeResearch 2013; 34:19-48). The leaky BRB potentiates the transport ofFGF21 from blood into retina. As local expression of FGF21 receptors hasbeen detected in total retina and in retinal neurons, circulating FGF21could directly act on retinal neurons to exert protective effects in DR.

In summary, there is an unmet need for the prevention and treatment ofDR. Maintaining retinal structure and function particularlyphotoreceptor activity improves retinal vascular stability, which can beachieved by two ways: i) modulating photoreceptor metabolism to matchthe energy supply; ii) slowing down the visual cycle to reduce theenergy demand.

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for treating or preventing neovascularization and/orangiogenesis in the eye of a subject, the method comprising: (a)identifying a subject having or at risk of neovascularization and/orangiogenesis in the eye; and (b) administering a pharmaceuticalcomposition comprising a stabilized fibroblast growth factor 21 (FGF21)agent to the subject in the eye of a subject
 2. (canceled)
 3. The methodof claim 1, wherein the subject has or is at risk of developing acondition selected from the group consisting of neovascular retinopathy,diabetic retinopathy in type I diabetes, retinopathy of prematurity(ROP), retinitis pigmentosa (RP) and macular telangiectasia (MacTel). 4.The method of claim 1, wherein neovascularization and/or angiogenesis inthe choroid or retinal cells of the eye is treated or prevented. 5.(canceled)
 6. The method of claim 1, wherein the stabilized FGF21 agentcomprises an FGF21 polypeptide or a modified FGF21 polypeptideconjugated to an antibody scaffold.
 7. (canceled)
 8. The method of claim6, wherein the modified FGF21 is dHis/Ala129Cys, or wherein the modifiedFGF21 is conjugated at Cys 129 to the antibody scaffold.
 9. The methodof claim 6, wherein two or more FGF21 polypeptide molecules areconjugated to one antibody scaffold.
 10. The method of claim 6, whereinthe antibody scaffold is a CovX-2000 scaffold.
 11. The method of claim1, wherein the stabilized FGF21 agent is a long acting FGF21 analog,comprising PF-05231023.
 12. The method of claim 1, wherein thestabilized FGF21 agent possesses a half-life of at least 1.5× thehalf-life of a native FGF21 peptide when assayed for stability underidentical conditions.
 13. The method of claim 1, wherein the stabilizedFGF21 agent possesses a half-life of at least 0.8 h in the circulationof a mammal, wherein the mammal is human.
 14. The method of claim 1,wherein the pharmaceutical composition is administered to the eye of thesubject.
 15. (canceled)
 16. A pharmaceutical composition for use intreating or preventing neovascularization, and/or angiogenesis in theeye of a subject comprising fibroblast growth factor 21 (FGF21), astabilized FGF21 agent, a modified FGF21 molecule, or combinationsthereof, and a pharmaceutically acceptable carrier.
 17. Thepharmaceutical composition of claim 16, wherein the stabilized FGF21agent comprises an FGF21 polypeptide or modified FGF21 polypeptideconjugated to an antibody scaffold.
 18. (canceled)
 19. Thepharmaceutical composition of claim 17, wherein the modified FGF21 isdHis/Ala129Cys, or wherein the modified FGF21 is conjugated at Cys 129to the antibody scaffold.
 20. The pharmaceutical composition of claim17, wherein two or more FGF21 polypeptide molecules are conjugated toone antibody scaffold.
 21. (canceled)
 22. The pharmaceutical compositionof claim 17, wherein the stabilized FGF21 agent is a long acting FGF21analog, comprising PF-05231023.
 23. (canceled)
 24. A method for treatingor preventing hyperglycemic retinopathy of prematurity (ROP) in asubject, the method comprising: (a) identifying a subject having or atrisk of hyperglycemic ROP; and (b) administering a pharmaceuticalcomposition comprising a fibroblast growth factor 21 (FGF21) agent to asubject, thereby treating or preventing hyperglycemic ROP in thesubject.
 25. (canceled) 26-30. (canceled)
 31. A method of treating orpreventing against photoreceptor dysfunction, inflammation and/ormorphology in a subject in need thereof, comprising administering to thesubject a composition comprising a therapeutically effective amount offibroblast growth factor 21 (FGF21) or a long-acting FGF21 analog.32-40. (canceled)
 41. A pharmaceutical composition comprising one ormore fibroblast growth factor 21 (FGF21) molecules, comprising:pegylated FGF21, modified FGF21 proteins, Fc-FGF21 fusion constructs,long acting FGF21 or combinations thereof.
 42. The pharmaceuticalcomposition of claim 41, wherein a pegylated FGF21 comprises an FGFR21with a R131AcF modification, coupled to PEG. 43-46. (canceled)